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One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG(1) molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature.

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.

Parkinson's disease is characterized by progressive degeneration of dopaminergic neurons. Thus the development of therapeutic neuroprotection and neurorescue strategies to mitigate disease progression is important. In this study we evaluated the neuroprotective/rescue effects of erythropoietin Fc fusion protein (EPO-Fc) and carbamylated erythropoietin Fe fusion protein (CEPO-Fc) in a rat model of Parkinson's disease. Adult female Sprague-Dawley rats received intraperitoneal injection of EPO-Fc, CEPO-Fc or PBS. Behavioral evaluations consisted of rota-rod, cylinder and amphetamine-induced rotation tests. In the neuroprotection experiment, the CEPO-Fc group demonstrated significant improvement compared with the EPO-Fc group on the amphetamine-induced rotation test throughout the four-week follow-up period. Histologically, significantly more tyrosine hydroxylase (TH)-positive neurons were recognized in the substantia nigra (SN) pars compacta in the CEPO-Fc group than in the PBS and EPO-Fc groups. In the neurorescue experiment, rats receiving CEPO-Fc showed significantly better behavioural scores than those receiving PBS. The histological data concerning striatum also showed that the CEPO-Fc group had significantly better preservation of TH-positive fibers compared to the PBS and EPO-Fc groups. Importantly, there were no increases in hematocrit or hemoglobin levels in the CEPO-Fc group in either the neuroprotection or the neurorescue experiments. In conclusion, the newly developed CEPO-Fc might confer neuroprotective and neurorescue benefits in a rat model of Parkinson's disease without the side effects associated with polycythemia. CEPO-Fc might be a therapeutic tool for patients with Parkinson's disease.

We recently developed a murine anti-idiotypic antibody (Ab2/3H6) versus the human monoclonal antibody 2F5, one of a few antibodies yet known to neutralize a broad range of HIV-1 primary isolates. Ab2/3H6 was not only able to bind to the paratope of mAb 2F5 but also significantly inhibited the binding of 2F5 to its synthetic epitope ELDKWA on gp41. In the present work we describe the partial humanization, expression, and characterization of Ab2/3H6 variants followed by several corresponding interaction studies with 2F5. The results of these studies support the high specificity of the recombinantly expressed Ab2s to the idiotype. Apparent affinities were designated by end point measurement and were similar compared to the murine Ab2/3H6. Moreover, the inhibition potency of chimeric Ab2/3H6 analyzed by in vitro studies could be shown to be the same as that detected for the hybridoma-derived murine Ab2/3H6.

For biotechnological research, development, and production various analytical methods are required to determine the quality of the target product. In this context, the determination of isoforms is state-of-the-art; however, the majority of applied techniques are more qualitative than quantitative. To address this fact, we evaluated different post- and pre-electrophoretic staining dyes for their applicability on linear IPG gels using recombinant human erythropoietin as a model protein. Each evaluated dyes was able to detect all isoforms reproducibly, but CyDyes were found to be the most promising. Using CyDyes, up to three samples can be focused on the same lane under identical electrophoretic conditions, thus, a fast, reproducible, sensitive and quantitative isoform determination can be performed. To illustrate the practical relevance, quantitative CyDye technique was used for the characterization of our model protein, recombinant human Epo-Fc. This method makes it possible to determine the isoform pattern of nonpurified supernatants as well as purified proteins. We conclude that quantitative pre-electrophoretic staining IEF using CyDyes is a fast, simple, accurate method to determine isoforms, which can be used in research, development, and manufacturing for product quality analysis, e.g., clone screening, process optimization, and purification monitoring.

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations.

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

Currently, one of the most used techniques for the determination of isoform pattern analysis is isoelectric focusing. Routinely, this is performed by immunoblotting. Blotting of proteins after isoelectric focusing on IPG gels may cause several problems, such as protein loss by the blotting itself and band broadening, in some cases the immunostaining with antibodies might be problematic. In the present study, an alternative isoform prestaining method with CyDye fluors is presented. For this approach, a highly glycosylated fusion protein, Epo-Fc, was used consisting of two recombinant human erythropoietin attached to the Fc part of a human IgG(1) molecule. By using CyDye fluors, up to three samples can be focused on the same lane under identical electrophoretic conditions. A fundamental benefit of this technique is the ability to perform quantitative isoform pattern analysis directly from serum-free culture supernatant.

The human monoclonal antibody 4E10 has been generated previously by immortalization of peripheral blood cells from an HIV-1-infected individual. This antibody binds to the linear epitope NWFDIT on gp41 and exhibits exceptional neutralizing activity against a broad spectrum of primary HIV-1 isolates. In the present study, molecular features, immunoreactivity, and functional activity of 4E10 were studied. The original hybridoma-derived 4E10 was of subtype IgG(3). Analysis of the variable segment of the heavy chain (VH) demonstrated extensive somatic mutations compared to the closest homologous germline gene VH1-69. Most amino acid substitutions occurred in the complementarity-determining region (CDR) 2, characteristic for an antigen-driven somatic maturation. The heavy chain of the CDR3 (H3) is of unusual length and cannot be attributed with certainty to any specific D(H) locus. To enable mass production and to prolong the in vivo half-life, 4E10 was subsequently cloned as IgG(1) in Chinese hamster ovary (CHO) cells. In additional studies, 4E10 was class switched to the IgM isotype. Binding to the linear epitope NWFDIT was not significantly changed after the cloning procedures. However, in vitro studies revealed dramatic differences in the neutralizing potential. The antiviral activity could be greatly enhanced by change of IgG(3) to IgG(1). In contrast, the IgM isotype almost completely lost its neutralizing potential.

It is still common practice to find the most suitable expression system for a given protein by trial and error, as there are no clear rules available to make this decision on the basis of the properties of the desired protein. This work discusses several decision points between bacteria and yeasts, namely Escherichia coli and Pichia pastoris. Based on the expression of human trypsinogen 1, advantages and disadvantages of both expression systems are discussed, emphasizing yields and the efforts to obtain a functional product. Briefly, in E. coli a high yield of insoluble product was obtained which required substantial efforts for resolubilisation, whereas with P. pastoris a functional product was obtained at a comparatively low yield.

The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA. A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation. As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.

Anti-idiotypic antibodies directed against potentially neutralizing anti-HIV-1 antibodies may mimic epitopes on gp41 otherwise cryptic to the immune system. This study reports the generation of murine monoclonal antibody Ab2/3H6 blocking the binding of human Ab1 2F5 to the synthetic epitope and to gp160 in an enzyme-linked immunosorbent competition assay. Ab2/2H6 diminished the neutralizing potency of 2F5 in an in-vitro neutralization assay. Ab2/3H6 Fab fragments were capable of inducing neutralizing immune and 2F5-specific responses in B6D2F1 mice applying a simple prime-boost regimen of immunization.

Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors. The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm. After cell lysis and harvesting by centrifugation, the inclusion bodies were solubilized in Zwittergent 3-14 solution and refolded by stepwise dilution. Specific alliin lyase activity could be recovered by this procedure.