Biochemical characterization and immunogenicity of Neureight, a recombinant full-length factor VIII produced by fed-batch process in disposable bioreactors.

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. Commercially available recombinant FVIII are produced using an expensive perfusion technology in stainless steel fermenters. A fed-batch fermentation technology was recently developed to produce 'Neureight', a full-length recombinant human FVIII, in Chinese hamster ovary (CHO) cells. Here, we investigated the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products, Helixate and Advate, produced in baby hamster kidney or CHO cells, respectively. Our results demonstrate the purity, stability and functional integrity of Neureight with a standard specific activity of 4235 ±â€¯556 IU/mg. The glycosylation and sulfation profiles of Neureight were similar to that of Advate, with the absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc, and with sulfation of Y1680, that is critical for FVIII binding to von Willebrand factor (VWF). The endocytosis of Neureight by human immature dendritic cells was inhibited by VWF, and its half-life in FVIII-deficient mice was similar to that of Advate, confirming unaltered binding to VWF. In vitro and in vivo assays indicated a similar immunogenicity for Neureight, Advate and Helixate. In conclusion, the production of full-length FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical, functional and immunogenic properties as products developed using the classical perfusion technology.

Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH).

Parathyroid hormone (PTH) is the primary regulator of serum calcium homeostasis and plays a major role in bone metabolism. Its actions are mediated via the PTH1 receptor (PTH1R) resulting in adenylate cyclase activation and consequently production of cyclic adenosine mono-phosphate (cAMP). The latter stimulates cellular metabolic pathways. This study describes the development, validation and applications of a novel cell-based potency assay for PTH using HEK293 cells over-expressing PTH1R. PTH concentration-dependent cAMP formation in these cells was quantitatively analyzed employing time-resolved fluorescence technology (TR-FRET). The optimized assay was precise, reproducible and exhibited a high sensitivity to PTH with a limit of quantification in the low picogram range. The potencies of differently manufactured PTH1-34 peptides, as well as a full-length variant (PTH1-84), were all accurately measured. Since PTH activity is inhibited by neutralizing antibodies against PTH, the assay was adapted to detect and measure neutralizing antibodies in human serum. Thus, applications of this novel cell-based PTH potency assay were extended to immunogenicity testing of PTH preparations in non-clinical and clinical settings.

Quantification of a pegylated interferon-alpha2a product by a customised and validated reverse phase-high performance liquid chromatography method.

There is increasing development of pegylated proteins as clinical products for therapeutic interventions in human disease. Quantification of such products relies on appropriately calibrated traditional methods, including reverse phase-high performance liquid chromatography (RP-HPLC). However, currently available pharmacopoeia calibrants, e.g., chemical reference substances (CRS), are highly purified non-pegylated proteins of known concentration. These are uncertain to be suitable for calibration purposes where the precise quantification of the mass of pegylated proteins, often heterogeneous with respect to polyethylene glycol (PEG) chain size, structure, attachment sites and isomer numbers and proportions, is required. In this study, a customised RP-HPLC method was developed and validated for the analysis of a pegylated IFN-α2a product having a linear 20kDa PEG chain (PEG20-IFN-α2a; Reiferon Retard(®)). Since the PEG20 moiety generated no signal at the detection wavelength of 210nm, the concentration of the base IFN-α2a molecules in PEG-IFN-α2a could be determined. By calculating the UV absorbance at 210nm of peak areas in their respective chromatographic profiles, a high correlation (r(2) ≥ 0.995) of PEG20-IFN-α2a concentrations with equal concentrations of the CRS of IFN-α2a, or of a well-characterised PEG20-IFN-α2a internal reference substance (IRS) was found. This finding confirmed the suitability of this CRS as a primary calibrant for mass determinations of PEG20-IFN-α2a by the customised RP-HPLC method. Application of this method to the quantitative analysis of 10 batches of Reiferon Retard(®) yielded accurate and consistent results, indicating its utility for mass determinations of current and future Reiferon Retard(®) batches.

Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha.

BACKGROUND: Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study's goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system. RESULTS: The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved. CONCLUSION: The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.