Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

INTRODUCTION: Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. AIMS: To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. METHODS AND RESULTS: The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. CONCLUSIONS: Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin.

The first recombinant human coagulation factor VIII of human origin: human cell line and manufacturing characteristics.

INTRODUCTION: Since the early 1990s, recombinant human clotting factor VIII (rhFVIII) produced in hamster cells has been available for haemophilia A treatment. However, the post-translational modifications of these proteins are not identical to those of native human FVIII, which may lead to immunogenic reactions and the development of inhibitors against rhFVIII. For the first time, rhFVIII produced in a human host cell line is available. AIM: We describe here the establishment of the first human production cell line for rhFVIII and the manufacturing process of this novel product. METHODS AND RESULTS: A human cell line expressing rhFVIII was derived from human embryonic kidney (HEK) 293 F cells transfected with an FVIII expression plasmid. No virus or virus-like particles could be detected following extensive testing. The stringently controlled production process is completely free from added materials of animal or human origin. Multistep purification employing a combination of filtration and chromatography steps ensures the efficient removal of impurities. Solvent/detergent treatment and a 20 nm pore size nanofiltration step, used for the first time in rhFVIII manufacturing, efficiently eliminate any hypothetically present viruses. In contrast to hamster cell-derived products, this rhFVIII product does not contain hamster-like epitopes, which might be expected to be immunogenic. CONCLUSIONS: HEK 293 F cells, whose parental cell line HEK 293 has been used by researchers for decades, are a suitable production cell line for rhFVIII and will help avoid immunogenic epitopes. A modern manufacturing process has been developed to ensure the highest level of purity and pathogen safety.

Large-scale preparation of thrombin from human plasma.

Thrombin was prepared from human blood plasma (batch size 1200 L). First, prothrombin was isolated by the following separation techniques: cryoprecipitation, ion-exchange chromatography (diethyl aminoethyl, DEAE-IEX), heparin affinity chromatography, a second DEAE-IEX step, and immobilized metal-affinity chromatography (IMAC). Prothrombin was then activated to thrombin, which was purified by hydrophobic interaction chromatography (HIC) and concentrated by ultrafiltration. This process is cost-effective because a waste fraction can be used from one of the steps (heparin affinity chromatography) in the commercial production of plasma-derived Factor IX (FIX). The final thrombin preparation had a purity of approximately 75% (specific activity approximately 2400 IU/mg protein), which is sufficient for its intended purpose in a fibrin glue. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Factor X (FX) activity analysis, and analytical HIC were also used to characterize the thrombin. Three substantially different techniques were used to reduce any viral activity, namely: solvent/detergent (S/D) treatment, pasteurization, and virus filtration (nanofiltration). The manufacturing process presented here would be suitable for large-scale production of thrombin with a high degree of virus safety.

Separation of monosaccharides by hydrophilic interaction chromatography with evaporative light scattering detection.

Hydrophilic interaction liquid chromatography (HILIC) was used to separate monosaccharides that are common in N-linked oligosaccharides in glycoproteins and other compounds. A TSKgel Amide-80 column was eluted with 82% acetonitrile, in 5 mM ammonium formate (pH 5.5). Column temperature was 60 degrees C and evaporative light scattering was used for detection (ELSD). With this method, L-fucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetylneuraminic acid, and D-glucuronic acid were separated, with detection limits of 0.3-0.5 microg for each monosaccharide, and intermediate precisions were 3-6% RSD (n=6).

Determination of triton X-100 in plasma-derived coagulation factor VIII and factor IX products by reversed-phase high-performance liquid chromatography.

Plasma protein pools are often virus-inactivated by the solvent-detergent method, using tri-n-butyl phosphate and Triton X-100, followed by removal and determination of these compounds. We used reversed-phase high-performance liquid chromatography for the determination of Triton X-100 in coagulation factor VIII and factor IX products, Octonativ-M and Nanotiv, respectively (Pharmacia, Stockholm, Sweden). The chromatographic system included a C18 silica column and a linear acetonitrile gradient. The advantage of this method is the low detection limit (0.3 microg/ml) combined with detection at 280 nm, which gives a more stable baseline and has less interference from other compounds. As compared to other methods, where shorter wavelengths are used.

Determination of the stabilizer sucrose in a plasma-derived antithrombin process solution by hydrophilic interaction chromatography with evaporative light-scattering detection.

Hydrophilic interaction chromatography (HILIC) is used for the quantitation of sucrose in the range of 10-100 micro g/mL. A poly-2-hydroxyethylaspartamide column is eluted with 25% water-75% acetonitrile, and evaporative light scattering is utilized for detection. A process sample of antithrombin (Atenativ) from Octapharma AB (Stockholm, Sweden) containing 20% sucrose is analyzed. The precision for this high-performance liquid chromatographic method is a percent relative standard deviation (%RSD) of 4, limit of detection (s/n=3) of 1 microg/mL, and mean recovery of spiked samples of 101% (RSD% of 3, n=6). Analysis time is 10 min/sample. Glucose, fructose, sodium citrate, sodium phosphate, Triton X-100, and tri-n-butyl phosphate do not interfere with the method.

Separation of latent, prelatent, and native forms of human antithrombin by heparin affinity high-performance liquid chromatography.

Latent antithrombin (LAT) is a partially denatured form of human antithrombin (AT). LAT does not inhibit clotting of the blood, but has previously been shown to inhibit angiogenesis and carcinogenesis. Another probably partially denatured form is the so-called prelatent AT (P-LAT), described by Larsson et al. [J. Biol. Chem. 276 (2001) 11996]. In the present work, an analytical heparin affinity chromatography method is described that separates an AT form, which is formed during the pasteurization process and which we believe to be identical to the previously described P-LAT, from native AT and LAT. Non-pasteurized AT was shown to contain no P-LAT, while four, heat-treated commercial AT products all contained P-LAT (1-6%, mean=4%). P-LAT has a slightly lower affinity to heparin than does native AT, but exhibits a much stronger heparin affinity when compared to LAT. P-LAT and native AT were shown to have very similar thrombin inhibiting activity, while LAT lacks such activity.

Separation between the alpha and beta forms of human antithrombin by hydroxyapatite high-performance liquid chromatography.

Human antithrombin (AT) inhibits several proteases in the coagulation system, including thrombin and factor Xa, and thus, plays an important role in the regulation of blood coagulation. The predominant form of AT in plasma is ATalpha, which contains four glycosylated asparagine residues, and the minor form is ATbeta, which lacks the Asn-135 glycosylation. In this study, hydroxyapatite high-performance liquid chromatography, using a segmented sodium phosphate gradient, was utilized for the high-resolution separation of ATalpha and ATbeta. The detection limit (signal-to-noise ratio of 3) for ATbeta was 30 microg/mL, corresponding to 0.5% of the injected concentration of AT. Two analyzed commercial AT products both contained about 2% ATbeta. This method is suitable for the determination of ATbeta in pure samples of native AT.

Preparative conversion of native human antithrombin to the latent form.

Human native antithrombin (AT) can be converted to a partially denaturated form of AT, known as latent AT (L-AT). This latent form of AT has been shown to exhibit strong antiangiogenic activity and also to suppress tumor growth in mice models. In the present work, a method is presented which induces the conversion of native AT to L-AT, using incubation at 60 degrees C, for 16 h, with 0.9 M ammonium sulfate, in 5mM Hepes buffer, pH 7.4, giving a recovery of more than 70%. L-AT was determined by integration of the low heparin affinity peak when analyzed by the affinity chromatography method. Native polyacrylamide gel electrophoresis was used to show that the preparation contained no aggregates. Hydrophobic interaction chromatography was also used for the separation of AT and L-AT.