Identification of CHO endogenous gene regulatory elements.

The objective of this approach was to identify new CHO endogenous gene regulatory elements that are capable of regulating foreign gene expression in recombinant CHO host cells. The standard technology for the production of many biopharmaceutical products is frequently based on expression vectors that utilize strong mammalian viral promoters like SV40 or CMV which allow for very high expression rates but this may lead to constitutive over-expression resulting in a permanent stress for the cell. In addition, some heterologous promoters are cell-cycle dependent and can be subject to gene silencing generating heterogeneity within the cell population.

Structure and basal transcription complex of RNA polymerase II core promoters in the mammalian genome: an overview.

The mammalian core promoter is a sophisticated and crucial component for the regulation of transcription mediated by the RNA polymerase II. It is generally defined as the minimal region of contiguous DNA sequence that is sufficient to accurately initiate a basal level of gene expression. The core promoter represents the ultimate target for nucleation of a functional pre-initiation complex composed of the RNA polymerase II and associated general transcription factors.

Plasmid-free T7-based Escherichia coli expression systems.

In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated.

Refolding of Npro fusion proteins.

The autoprotease Npro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C-terminus releasing the target protein with its authentic N-terminus. Npro wild-type and its mutant EDDIE, respectively, were fused N-terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence-evasion-factor, and the short 16 amino acid peptide pep6His.

High-throughput system for determining dissolution kinetics of inclusion bodies.

Efficient solubilization is a crucial step during inclusion body processing and dissolving conditions were usually empirically established. Here we describe a new methodology for rapid screening of solubilization conditions and evaluation of dissolution kinetics in microtiter plates. Increase of protein in solution over time was directly related to decrease of turbidity measured by absorbance at 600 nm.

A novel Ecotin-Ubiquitin-Tag (ECUT) for efficient, soluble peptide production in the periplasm of Escherichia coli.

Background: Many protocols for recombinant production of peptides and proteins include secretion into the periplasmic space of Escherichia coli, as they may not properly fold in the cytoplasm. If a signal peptide is not sufficient for translocation, a larger secretion moiety can instead be fused to the gene of interest. However, due to the covalent linkage of the proteins, a protease recognition site needs to be introduced in between, altering the N-terminus of the product.

Identification of transgene integration loci of different highly expressing recombinant CHO cell lines by FISH.

Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of site-directed integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa-Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities.

Identification of CHO endogenous promoter elements based on a genomic library approach.

Today the standard technology for the production of many biopharmaceutical products from mammalian cell systems is frequently based on expression vectors that utilize strong mammalian active viral promoters like CMV or SV40 for driving recombinant gene transcription. On one hand these promoters allow very high expression rates, but on the other hand, they can lead to constitutive over-expression of the gene of interest resulting in a permanent stress on the cell. Another drawback is that they are cell cycle-dependent and can be subject to gene silencing which leads to a heterogeneity within the cell population.

Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies.

Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses.

N(pro) fusion technology to produce proteins with authentic N termini in E. coli.

We describe a prokaryotic expression system using the autoproteolytic function of N(pro) from classical swine fever virus. Proteins or peptides expressed as N(pro) fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus.

Applicability of different fluorescent dyes for isoform quantification on linear IPG gels.

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.

Characterisation of recombinant CHO cell lines by investigation of protein productivities and genetic parameters.

We have generated a recombinant CHO cell line expressing the fusion protein EpoFc. After selection and screening, protein expression, gene and mRNA copy numbers were analysed in order to gain more information on the influence of genetic parameters on the productivity and stability of production cells. Results from semi-quantitative blot methods were compared to quantitative PCR (qPCR) analyses, whose advantage mainly lies in their higher sensitivity, and the cheaper and faster methodology.

Current status of technical protein refolding.

The expression of heterologous proteins in microbial hosts frequently leads to the formation of insoluble aggregates. To fully exploit the production capacity of the cells, efficient strategies for further processing have to be developed. While in lab scale matrix assisted refolding techniques, especially of histidine-tagged proteins have become very popular, in production scale refolding by dilution is still predominant due to its simplicity.

Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters.

Chromatographic and electrophoretic characterization of protein variants.

Almost all proteins are expressed in several variants, also known as isoforms. Individual protein variants differ by modifications of the individual amino acid side chains, or the N- or C-terminus. Typical modifications are glycosylation, phosphorylation, acetylation, methylation, deamidation or oxidation.

Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells.

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.

A novel strategy for quantitative isoform detection directly performed from culture supernatant.

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 situ determination of adsorption kinetics of proteins in a finite bath.

A method for fast in situ measurement of adsorption kinetics based on a finite bath was developed. We modified the conventional finite bath by replacing the external loop by a dip probe which enables in situ measurement of the concentration change in the contactor. Deposition of adsorbent particles on the reflection surface of the dip probe compromised measurements.

Engineering protein A affinity chromatography.

Staphylococcal protein A can selectively interact with immunoglobulins. This protein is widely used as a ligand for affinity chromatography to purify therapeutic antibodies on an industrial scale. This type of affinity chromatography constitutes a generic step in processing antibodies.