Marina Etcheverrigaray

Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality.

We have demonstrated that temperature reduction from 37 to 33 °C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO‐K1‐hGM‐CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO‐K1‐hGM‐CSF cells were cultured in a biphasic mode: first, a 37 °C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 °C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 °C. Furthermore, the total rhGM‐CSF production increased 6 times in the culture shifted to 33 °C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N‐ and O‐glycosylation of the protein produced under both cell culture conditions using high‐pH anion‐exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM‐CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO‐K1‐hGM‐CSF cells without affecting product quality, representing a novel procedure for the rhGM‐CSF production process.

Suspension-Vero cell cultures as a platform for viral vaccine production

Since Vero cells are currently considered as an acceptable cell substrate to produce a wide range of viruses, we developed a virus production platform using Vero cells adapted to grow in suspension in serum-free media. After adapting anchorage-dependent Vero cells to grow as a free-cell suspension, vesicular stomatitis virus, herpes simplex virus 1 and polio virus 1 production rates were evaluated in batch cultures using spinner flasks and perfused cultures in a bioreactor. The achieved results constitute valuable information for the development of a low-cost high-productivity process using a suspension culture of Vero cells to produce viral vaccines.

Highly glycosylated human alpha interferon: An insight into a new therapeutic candidate.

The type I human interferon alpha (hIFN-alpha) family consists of small proteins that exert a multiplicity of biological actions including antiviral, antiproliferative and immunomodulatory effects. However, though administration of recombinant hIFN-alpha2b is the current treatment for chronic hepatitis B and C and for some types of cancers, therapy outcomes have not been completely satisfactory. The short serum half-life and rapid clearance of the cytokine accounts for its low in vivo biological activity. Here we describe and characterize a long-acting rhIFN-alpha2b mutein, 4N-IFN, which has been created by introducing four N-glycosylation sites via site-directed mutagenesis. The hyperglycosylated protein was found to have a 25-fold longer plasma half-life than the non-glycosylated rhIFN-alpha2b, even greater than the commercial pegylated derivative Intron-A PEG. In addition, glycosylation increased the in vitro stability of the mutein against serum protease inactivation. Interestingly, despite its lower in vitro activity, 4N-IFN showed a markedly enhanced in vivo antitumor activity in human prostate carcinoma implanted in nude mice. MALDI-TOF MS and HPAEC-PAD carbohydrate analyses revealed the presence of high amounts of tetrasialylated (40%) and trisialylated (28%) N-glycan structures, which are consequently responsible for the improved characteristics of the cytokine, making 4N-IFN a new therapeutic candidate for viral and malignant diseases.

Influence of carbohydrates on the stability and structure of a hyperglycosylated human interferon alpha mutein

Protein physical and chemical instability is one of the major challenges in the development of biopharmaceuticals during every step of the process, ranging from production to final delivery. This is particularly applicable to human recombinant interferon alpha-2b (rhIFN-alpha2b), a pleiotropic cytokine currently used worldwide for the treatment of various cancer and chronic viral diseases, which presents a poor stability in solution. In previous studies, we have demonstrated that the introduction of four N-glycosylation sites in order to construct a heavily glycosylated IFN variant (4N-IFN) resulted in a markedly prolonged plasma half-life which was reflected in an enhanced therapeutic activity in mice in comparison with the commercial non-glycosylated rhIFN-alpha2b (NG-IFN). Herein, we evaluated the influence of glycosylation on the in vitro stability of 4N-IFN towards different environmental conditions. Interestingly, the hyperglycosylated cytokine showed enhanced stability against thermal stress, acid pH and repetitive freeze-thawing cycles in comparison with NG-IFN. Besides, microcalorimetric analysis indicated a much higher melting temperature of 4N-IFN, also demonstrating a higher solubility of this variant as denoted by the absence of precipitation at the end of the experiment, in contrast with the NG-IFN behaviour. Furthermore, far-UV circular dichroism (CD) spectrum of 4N-IFN was virtually superimposed with that of NG-IFN, indicating that the IFN structure was not altered by the addition of carbohydrate moieties. The same conclusion could be inferred from limited proteolysis studies. Our results suggest that glycoengineering could be a useful strategy for protecting rhIFN-alpha2b from inactivation by various external factors and for overcoming aggregation problems during the production process and storage.

Choice of the adequate quantification method for recombinant human GM-CSF produced in different host systems

Three enzyme-linked-immunosorbent assays (ELISA) were developed and compared with a bioassay to quantify the recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF). These assays were suitable to quantify the non-glycosylated rhGM-CSF present in mixtures with variable protein content, and therefore useful for determining concentrations of the cytokine in production processes. Among these assays, the competitive ELISA, developed with a MAb that recognises an epitope not involved in glycosylation, was the only one suitable to quantify the glycosylated form of rhGM-CSF produced in mammalian cell cultures.

Rabies virus-like particles expressed in HEK293 cells

Rabies is an infectious viral disease with a mortality rate close to 100%. Currently, there is a need to generate cheaper and more immunogenic vaccines for the effective prevention of rabies, mostly in developing countries. Virus-like particles have been widely used in viral vaccine production due to their high immunogenicity and safety during the production process. Rabies virus glycoprotein is the major antigen to trigger a protective immune response and the only protein capable of generating virus neutralizing antibodies. In this study we describe the development of a recombinant stable cell line for the production of rabies virus-like particles (RV-VLPs) expressing the rabies virus glycoprotein by lentivirus-based transduction of HEK293 cells. Protein expression was analyzed by flow cytometry, fluorescence microscopy, western blot and ELISA. Particles were purified from culture supernatant and their size and morphology were studied. Furthermore, mice were immunized with RV-VLPs, formulated with adjuvant, and these particles were able to produce a specific antibody response, demonstrating that these virus-like particles present a promising rabies vaccine candidate.

A scFv antibody fragment as a therapeutic candidate to neutralize a broad diversity of human IFN-alpha subtypes

Despite their significant role in maintaining the normal physiology, cytokines may cause pathological conditions when they are overproduced. In this way, the increased expression of human interferon alpha (hIFN-alpha) is associated with acute viral infections, inflammatory disorders and several autoimmune illnesses, where the cytokine may be a factor in either initiating or maintaining the disease. Currently, there are several mAbs marketed for a variety of indications and many more in clinical trials, in which IFN-alpha represents a potential target for antibody-based therapy. A panel of 11 murine mAbs was prepared using recombinant hIFN-alpha2b as immunogen, all of which bound to the native form of the cytokine with affinity constants ranging from 1.7x10(7) M(-1) to 1.4x10(10) M(-1). An epitope mapping protocol demonstrated four spatially distinct areas of the protein recognized by the mAbs. Taking into account the characterization of the antibodies and their ability to inhibit the IFN-alpha biological activity, four mAbs were selected to produce scFv fragments. One of these fragments (CA5E6) was able to neutralize a wide spectrum of subtypes of the IFN-alpha family, including the recombinant cytokines hIFN-alpha2a and hIFN-alpha2b and a heterogeneous collection of IFN-alpha produced by activated leukocytes and Namalwa cells. With the aim of improving the affinity of the selected fragment, a standard error-prone PCR method was carried out. By using this strategy, it was possible to generate a new fragment (EP18) with increased affinity and ability to neutralize a broad diversity of IFN-alpha subtypes. Consequently, the scFv EP18 represents a potential therapeutic agent for those immune and inflammatory diseases which are associated with an increased IFN-alpha expression.

Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero)

Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines. We generated a Vero cell line adapted to grow in suspension (sVero) in a serum-free medium and evaluated it for its potential as host cell for influenza vaccine production. Initially we studied the capacity of sVero cells to grow in the presence of incremental concentrations of trypsin. In comparison with adherent Vero cells (aVero), we found that sVero cells maintain their growth kinetics even with a three-fold increase in trypsin concentration. The influence of the conditions of infection on the yield of H1N1 produced in serum-free suspension cultures of sVero cells was investigated by a 2(2) full factorial experiment with center point. Each experiment tested the influence of the multiplicity of infection (m.o.i.) and trypsin concentration, on production yields at two levels, in four possible combinations of levels and conditions, plus a further combination in which each condition was set in the middle of its extreme levels. On the basis of software analysis, a combination of m.o.i. of 0.0066TCID(50%)/cell and trypsin concentration of 5μg/1.0×10(6) cells with a desirability of 0.737 was selected as the optimized condition for H1N1 production in sVero cells. Our results show the importance of proper selection of infection conditions for H1N1 production on sVero cells in serum-free medium.

A strategy to obtain recombinant cell lines with high expression levels. Lentiviral vector-mediated transgenesis

BackgroundThe primary goal of any recombinant protein produc-tion is to achieve successful gene transfer and expres-sion in a target cell. There are two general categories ofdelivery vehicles/vectors employed in protein expressionprotocols. The first category includes the non-viral vec-tors, ranging from direct injection of DNA to complex-ing DNA with cationc lipds, polylysine, etc. The secondcategory comprises DNA and RNA viral vectors.Viruses have evolved specific mechanism to delivertheir genetic material to target cell nuclei. Virus mem-bers of family Retroviridae, e.g. retroviruses and lenti-viruses, are among the most widely used viral vectors.The use of lentiviral vectors has been increasing becausethe vector system has attractive features. Lentiviruseshave an advantage over retroviruses in that they caninfect both dividing and non-dividing cells and thereforehave attracted much attention regarding the potential asvectors for gene delivery/therapy. Once integrated intothe genome, recombinant cell lines are selected usingdifferent selection mechanisms.ResultsLentivirus particles were produced by simultaneous co-transfection of HEK 293T cells with four plasmids. Thepackaging construct (pMDLg/pRRE) [1], the VSV-G-expressing construct (pMD.G) [2], the Rev-expressingconstruct (pRSV-Rev) [1], and the self-inactivating (SIN)lentiviral vector construct containing the green fluores-cent protein (GFP) reporter gene (pLV-PLK-eGFP). Themedium containing lentiviral particles was collected 48h after transfection, clarified by centrifugation 10 min at2000 rpm and then stored at -80°C. To determine viraltiters, HEK 293T cells were seeded at 3 x 10

Novel chemometric strategy based on the application of artificial neural networks to crossed mixture design for the improvement of recombinant protein production in continuous culture

The optimal blends of six compounds that should be present in culture media used in recombinant protein production were determined by means of artificial neural networks (ANN) coupled with crossed mixture experimental design. This combination constitutes a novel approach to develop a medium for cultivating genetically engineered mammalian cells. The compounds were collected in two mixtures of three elements each, and the experimental space was determined by a crossed mixture design. Empirical data from 51 experimental units were used in a multiresponse analysis to train artificial neural networks which satisfy different requirements, in order to define two new culture media (Medium 1 and Medium 2) to be used in a continuous biopharmaceutical production process. These media were tested in a bioreactor to produce a recombinant protein in CHO cells. Remarkably, for both predicted media all responses satisfied the predefined goals pursued during the analysis, except in the case of the specific growth rate (mu) observed for Medium 1. ANN analysis proved to be a suitable methodology to be used when dealing with complex experimental designs, as frequently occurs in the optimization of production processes in the biotechnology area. The present work is a new example of the use of ANN for the resolution of a complex, real life system, successfully employed in the context of a biopharmaceutical production process.