Publications

Highlights • Therapeutic factor VIII (FVIII), used to treat bleedings in patients with hemophilia A, is one of the most expensive drugs on the market. • A cost-optimized fed-batch fermentation technology in single-use bioreactors was recently developed to produce ‘Neureight’, a full-length recombinant human FVIII. • Here, we confirm the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products. Abstract Hemophilia a is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. The 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±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.

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein consisting of noncovalently linked α and b subunits. It stimulates the growth of immature follicles in the ovary and primary spermatocytes in the testis and thus plays an important role in the human reproduction process. Human menopausal gonadotropin for infertility treatment was first introduced into clinical practice in 1950. Subsequently, treatments with urinary FSH have been replaced by recombinant human FSH (rh-FSH), which has been shown to provide several advantages such as absence of luteinizing hormone activity, higher specific activity, and lower risk of infection. The first commercially marketed rh-FSH was developed using a stainless steel vessel and a continuous perfusion system with serum containing media. However, many biopharmaceutical companies are switching from such production systems to single-use (SU) bioreactors. This has been due to a wealth of manufacturing advantages of SU bioreactors, including fewer validation requirements, lower contamination risks, elimination of cleaning and sterilization steps, and rapid turnaround between batches, which leads to reduced preparation times. Such advantages have contributed to reduced capital cost by 40–50%, operating costs by 20–30%, and time to-build by 30% when compared with conventional stainless steel vessel technology. Using stably transformed Chinese hamster ovary (CHO) cells secreting rh-FSH, we describe our company’s pure recombinant human gonadotropin process (Gonapure) for consistently high hormone production in an upstream fed-batch process to final 50-L and 200-L SU bioreactor scales, followed by and efficient, robust downstream purification pathway. We also discuss the cost benefits of our manufacturing strategy.

tParathyroid hormone (PTH) is the primary regulator of serum calcium homeostasis and plays a majorrole in bone metabolism. Its actions are mediated via the PTH1 receptor (PTH1R) resulting in adenylatecyclase activation and consequently production of cyclic adenosine mono-phosphate (cAMP). The latterstimulates cellular metabolic pathways. This study describes the development, validation and appli-cations of a novel cell-based potency assay for PTH using HEK293 cells over-expressing PTH1R. PTHconcentration-dependent cAMP formation in these cells was quantitatively analyzed employing time-resolved fluorescence technology (TR-FRET). The optimized assay was precise, reproducible and exhibiteda high sensitivity to PTH with a limit of quantification in the low picogram range. The potencies of dif-ferently manufactured PTH1–34peptides, as well as a full-length variant (PTH1–84), were all accuratelymeasured. Since PTH activity is inhibited by neutralizing antibodies against PTH, the assay was adaptedto 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-clinicaland clinical settings.

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 nonpegylated 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-alpha 2a product having a linear 20 kDa PEG chain (PEG20-IFN-alpha 2a; Reiferon Retard®). Since the PEG20 moiety generated no signal at the detection wavelength of 210 nm, the concentration of the base IFN-alpha 2a molecules in PEG-IFN-alpha 2a could be determined. By calculating the UV absorbance at 210 nm of peak areas in their respective chromatographic profiles, a high correlation (r2 ≥ 0.995) of PEG20-IFN-alpha 2a concentrations with equal concentrations of the CRS of IFN-alpha 2a, or of a well-characterised PEG20-IFN-alpha2a internal reference substance (IRS) was found. This finding confirmed the suitability of this CRS as a primary calibrant for mass determinations of PEG20-IFN-alpha 2a 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.

Background: Osteoporosis is a bone disease of the elderly that leads to increased risk of fracture. Currently, it affects more than 200 million adults worldwide, placing an enormous economic burden on healthcare providers. Present approved drug treatments of osteoporosis, for example, using bisphosphonates, only reduce bone mineral loss, are variable in prevention of future fractures and can induce serious complications long term. From 2002, availability of the recombinant active, N-terminal 1-34 fragment of human parathyroid hormone 1-34, which directly stimulates bone formation, has provided an alternative, potentially more effective therapeutic agent for treating osteoporosis, especially in its most severe form. We have adopted the methylotrophic yeast strain Hansenula polymorpha (H. polymorpha) for production of recombinant parathyroid hormone (rPTH) 1-34 since its expression system is highly inducible and target proteins are secreted intact into the fermentation broth without the need for enzymatic cleavage of a fusion protein. Methods: Culture conditions for the growth of selected clones of H. polymorpha super-transformants and their rPTH production capacity were sequentially optimized through the microtitre plate, shake flask, small tank bioreactor scales to the large bioreactor scale. Results: The production of rPTH 1-34 by transformed H. polymorpha was greatly enhanced by adding the calnexin chaperone gene to the expression plasmid. Implementation of final optimized culture medium constituents and operational fermentation conditions, of which maintenance of constant oxygen tension (pO2) was found to be a critical factor at the 80 litre scale, resulted in yields up to 150 mg active rPTH 1-34 per liter of culture supernatant. Conclusion: A robust fermentation process for the efficient production of active rPTH 1-34 based on a calnexin super-transformed H. polymorpha clone was developed. Potentially, this has cost-saving benefits for the production and marketing of clinical-grade rPTH 1-34.

Current downstream processing strategies for recombinant proteins often require multiple chromatographic steps, which may lead to poor overall yields. Product purification can be especially difficult when a target protein displays reduced stability, forms isoforms or misprocessed variants, or needs to be purified from a complex mixture containing a high degree of contaminants. One technology that has been developed to tackle such limitations is based on custom-made chromatography matrices containing camelid-based single-domain antibody fragments. With a molecular weight of only 12–15 kDa, these fragments are significantly smaller than conventional antibodies (with molecular weights 150–160 kDa).

Abstract 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. Keywords: Staphylokinase, Hansenula polymorpha, Recombinant protein, Fermentation, Scale-up, HTP

The availability of robust disposable fermentation systems has significantly grown over the last years and gains increasing popularity due to cost pressure and changing requirements for bioreactors in the biopharmaceutical industry. Cost reduction of about 30% compared to traditional stainless steel fermentation systems, reduced change-over times, higher flexibility and lower contamination risks are some of the key advantages. As the disposable fermentation technology is relatively young, there is no approved production process for a recombinant protein established to date. As shown in Figure 1, we present the first cultivation process for a chinese hamster ovary (CHO) derived recombinant human follicle stimulating hormone (rh-FSH) based on a complete disposable upstream pathway – from cell revitalization up to a 50 L cultivation scale [Vial - T-flask – 1 L SuperSpinner D1000 – 10 L Biostat Cultibag (WAVE) – 50 L HyClone SUB].

The use of disposables in downstream processes is well established in terms of different filtration technologies, buffer preparation and storage of liquids and became increasingly popular over the last years. Disposable systems are generally known to have several significant advantages in comparison to conventional concepts such as risk reduction for cross contamination. Disposables also save costs, reduce buffer consumption, labor and capital space and minimize time of cleaning and cleaning validation of approximately 30%. Recently, a number of new technologies for single use chromatography systems attracted many applicants to integrate them in downstream process strategies. We explored disposable chromatography as a capture and/or intermediate step [Sartobind® Q and Sartobind® Phenyl] in a defined purification process of r-h FSH. We herein compare the conventional chromatography pathway (P1) and alternative pathways which include one or two disposable chromatography steps (P2 and P3 ) in respect to their elution profile, yield and purity of the target protein. Results were found to be comparable with several advantages of the disposable membrane adsorber, in addition to the above mentioned benefits; the disposable membrane adsorber solved a carryover problem monitored with the traditional chromatography in the capture step.

Worldwide, most cases of acute hepatitis are caused by viruses. Hepatitis B (HBV) and C (HCV) viruses, in addition have the potential to cause chronic liver damage. Formerly known as the etiological agent of non-A, non-B hepatitis, HCV was identified in 1989 [1], but, despite the knowledge of its biological properties, no vaccine currently exists. HCV is endemic to most regions of the world, though prevalence rates vary widely. Nowadays over 170 million people are infected by HCV (about 3% of the global population). The majority of infections are clustered in developing nations, particularly in Asia and Africa. Liver disease (mostly due to HBV and HCV infections) accounts for 1.6% of the global burden of diseases, which translates into the loss of as many as 4.2 million Disability- Adjusted Life Years (DALYs) per year, a toll that is comparable to that of colorectal cancers (4.6 million) and breast cancer (4.2 million). Thus, the medical and economic consequences of liver diseases are extensive, particularly in developing countries, where incomes are lower and prevalence rates are higher. HCV accounts for a sizable proportion of cases of chronic liver disease, liver disease deaths, and hepatocellular carcinoma and represents the most common indication for liver transplantation. Projections based on the current prevalence of infection and anticipated rates of progression suggest that morbidity and mortality, as well as the medical care costs attributable to HCV infection will escalate alarmingly during the next two decades [2]. Six major genotypes (1 to 6), and more than 50 subtypes of HCV have been described so far [3]. Treatment is not indicated in all cases since several factors, including the very high cost of treatment, its long duration and its significant and unpleasant side effects should be taken into account. Chronically-infected patients are first tested for elevated liver enzymes, which indicate stress on the liver. Where available, patients with high enzymes can be tested for fibrosis using Fibrotest [4] and Fibroscan [5], two non-invasive methods. In case of discordant results, or unavailability of these tools due to high expense or other reasons, the diagnosis must be confirmed by liver biopsy, an invasive technique. Currently, histological staging of liver biopsies is the most reliable predictor of prognosis and the likelihood of disease progression for HCV. Patients suffering from liver damage must then be evaluated for contraindications to treatment, including pregnancy, uncontrolled mood disorders, etc. Patients deemed eligible for treatment for chronic HCV follow a 24- to 48-week course (depending on genotype) of Pegylated interferon and ribavirin. Sustained Virologic Response (SVR) is achieved in approximately 40% of patients with genotype 1, 60% of patients with genotype 4, and 80% of patients with genotypes 2 and 3. Treatment failure can be quite accurately predicted within 4 weeks of initiation treatment, since patients who experience a Rapid Virologic Response (RVR) respond, in almost all cases, to treatment later on. Acute HCV infections are rarely caught in time, but can be treated with 12–24 weeks of Pegylated interferon with or without ribavirin [6]. However, it is strongly recommended not to treat just after exposure before the appearance of markers of HCV replication. It is recommended that treatment should be offered within the first 12 weeks of diagnosis (in case of positive PCR and elevation of ALT) to offer the best chance to obtain a SVR.

Introduction: Isaacs and Lindenmann, who studied what had been a subject for research since 1935, i.e. the phenomenon of viral interference, reported in 1957 a factor that conferred the property of viral interference and coined the name interferon (IFN) to describe this new substance. In the following decades many attempts were undertaken in order to purify IFN to homogeneity. As a result, IFN was found to be heterogeneous, comprising a family of distinct, but structurally related, IFN molecules. Today these are classified as type I, all of which bind to the same receptor consisting of IFNAR-1 and -2. A single antigenically distinct type II IFN has also been characterized. As a response to stimulation by a various pathogens, type I IFNs are produced and secreted by virtually all eukaryotic cells. They can be mass produced by biotechnological processes, and both natural and recombinant IFNs have received regulatory approval in many countries for the treatment of diseases, including chronic hepatitis (CH), malignant melanoma, non-Hodgkin’s lymphoma (NHL), chronic myelogenous leukemia (CML), multiple sclerosis (MS) and others.