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.

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.

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.

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.

Improved expression of recombinant human factor IX by co-expression of GGCX, VKOR and furin.

Recombinant human FIX concentrates (rhFIX) are essential in the treatment and prevention of bleeding in the bleeding disorder haemophilia B. However, due to the complex nature of FIX production yields are low which leads to high treatment costs. Here we report the production of rhFIX with substantially higher yield by co-expressing human FIX with GGCX (γ-glutamyl carboxylase), VKOR (vitamin K epoxide reductase) and furin (paired basic amino acid cleaving enzyme) in Chinese hamster ovary (CHO) cells. Our results show that controlled co-expression of GGCX with FIX is critical to obtain high rhFIX titre, and, that co-expression of VKOR further increased the yield of active rhFIX. Furin co-expression improved processing of the leader peptide of rhFIX but had a minor effect on yield of active rhFIX. The optimal expression level of GGCX was surprisingly low and required unusual engineering of expression vector elements. For VKOR and furin the control of expression was less critical and could be achieved by standard vector element. Using our expression vectors an rhFIX-producing clone with an expression level of up to 30 mg/L of active rhFIX was obtained. In addition an efficient single step purification method was developed to obtain pure and active rhFIX with up to 94% yield.

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

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

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.

Pilot production of recombinant human clotting factor IX from transgenic sow milk.

Valuable pharmaceutical proteins produced from the mammary glands of transgenic livestock have potential use in the biomedical industry. In this study, recombinant human clotting factor IX (rhFIX) produced from transgenic sow milk for preclinical animal studies have been established. The transgenic sow milk was skimmed and treated with sodium phosphate buffer to remove abundant casein protein. Then, the γ-carboxylated rhFIX fraction was segregated through the Q Sepharose chromatography from uncarboxylated one. For safety issue, the process included virus inactivation by solvent/detergent (S/D) treatment. Subsequently, the S/D treated sample was loaded into the Heparin Sepharose column to recover the rhFIX fraction, which was then reapplied to the Heparin Sepharose column to enhance rhFIX purity and lower the ratio of activated form rhFIX (rhFIXa) easily. This was possible due to the higher affinity of the Heparin affinity sorbent for rhFIXa than for the rhFIX zymogen. Furthermore, an IgA removal column was used to eliminate porcine IgA in purified rhFIX. Finally, nanofiltration was performed for viral clearance. Consequently, a high-quality rhFIX product was produced (approximately 700 mg per batch). Other values for final rhFIX preparation were as follows: purity, >99%; average specific activity, 415.6±57.7 IU/mL and total milk impurity, <0.5 ng/mg. This is the first report that described the whole process and stable production of bioactive rhFIX from transgenic sow milk. The overall manufacturing process presented here has the potential for industrial production of rhFIX for treatment of hemophilia B patients.

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.

Use of proteomics for validation of the isolation process of clotting factor IX from human plasma.

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

Half-life extension through albumin fusion technologies.

Haemophilia B is characterized by a deficiency of coagulation factor IX (FIX), a protein that is important in the process of haemostasis and normal blood clotting. Recurrent bleeding into joints and soft tissues is the hallmark of severe haemophilia B. The goal of treatment is to prevent and manage haemorrhage and thereby reduce disabling joint and tissue damage, improve quality of life, and extend life expectancy. Current treatment with FIX replacement concentrates often requires repeated, frequent infusions, owing to the relatively short terminal half-life of FIX in the circulation. We have developed a unique technology for improving the biological characteristics of FIX in vivo. For this approach, recombinant FIX (rFIX) was genetically fused to albumin via a cleavable peptide linker. Constructs of the fusion protein were expressed in mammalian cells and characterized following purification. In vitro activation studies demonstrated that cleavage of the linker occurred in parallel with FIX activation. The molar specific clotting activity of the cleavable fusion protein (rIX-FP) was 10- to 30-fold greater than that of the fusion protein with non-cleavable linkers. In rats, rabbits, and FIX-deficient mice, the pharmacokinetics of rIX-FP were significantly improved compared with rFIX. Using the tail-clip bleeding model in FIX-deficient mice, rIX-FP effectively corrected the bleeding time and blood loss. These findings suggest that rIX-FP may be a promising therapy for the treatment of patients with haemophilia B.

Secondary prophylaxis with factor IX concentrates: continuous infusion.

Haemophilia patients may require prolonged treatment during peri-operative period or life-threatening bleedings. Intermittent bolus infusions of factor concentrates have been used successfully for many years. However, pharmacokinetics may vary among products and patients, and the wide fluctuations in factor levels during therapy can make management inaccurate and unsafe. Specific protocols for continuous infusion have been developed, which may decrease factor utilization, facilitate laboratory monitoring of factor levels, and may decrease the overall cost of therapy. Continuous infusion of different clotting factor concentrates (factor VIII, factor IX, porcine factor VIII, rFVIIa, and activated prothrombin complex concentrates) has been associated with excellent haemostasis and safety: indeed, by continuous infusion unsafe low troughs or dangerous high concentrations can be avoided. In this review, the pharmacokinetic aspects of the continuous infusion of plasma-derived and recombinant factor IX concentrates are showed. Finally, the advantages and disadvantages of continuous infusion are discussed.

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.

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.

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.

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

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

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.