Laupheim, Germany
Laupheim, Germany

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Sandhu U.,Helmholtz Center for Infection Research | Cebula M.,Helmholtz Center for Infection Research | Behme S.,Helmholtz Center for Infection Research | Riemer P.,Helmholtz Center for Infection Research | And 8 more authors.
Nucleic Acids Research | Year: 2011

Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreERT2, and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreERT2. The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts. © 2010 The Author(s).


PubMed | Biberach University of Applied Sciences, Rentschler Biotechnologie GmbH and University of Ulm
Type: Journal Article | Journal: Biotechnology and bioengineering | Year: 2016

Cell engineering and bioprocess optimizations such as low temperature cultivation represent powerful tools to improve cellular performance and product yields of mammalian production cells. Besides monoclonal antibodies (mABs), novel biotherapeutic formats such as viral vectors will gain increasing importance. Here, we demonstrate that similar to Chinese hamster ovary (CHO) cells, product yields of recombinant adeno-associated virus (rAAV) producing HeLa cells can be markedly increased by low temperature cultivation. MicroRNAs (miRNAs) are small non-coding RNAs that critically regulate cell phenotypes. We thus investigated differential miRNA expression in response to mild hypothermia in CHO and HeLa production cells. We discovered miR-483 to be substantially up-regulated upon temperature down-shift in both cell types. Functional validation experiments revealed that introduction of miR-483 mimics led to a significant increase in both rAAV and mAB production in HeLa and CHO cells, respectively. Furthermore, inhibition of miR-483 up-regulation during mild hypothermia significantly decreased product yields, suggesting that miR-483 is a key regulator of cellular productivity in mammalian cells. In addition, miRNA target gene identification indicated that miR-483 might regulate genes directly involved in cellular survival and protein expression. Our results highlight that miR-483 is a valuable tool for product-independent engineering of mammalian production cells.


PubMed | McGill University, National Research Council Canada, Rentschler Biotechnologie GmbH and University of Ulm
Type: Journal Article | Journal: Biotechnology journal | Year: 2016

Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing.


Eiberle M.K.,Boehringer Ingelheim | Eiberle M.K.,Rentschler Biotechnologie GmbH | Jungbauer A.,University of Natural Resources and Life Sciences, Vienna
Biotechnology Journal | Year: 2010

Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features industrially. However, the value of an inclusion body-based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies that are economical and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein into its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, the yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (≤ 0.1 mg/mL). However, large buffer tanks and time-consuming concentration steps are required. We attempt to answer the question of the extent to which refolding of proteins is protected by patents. Low-molecular mass additives have been developed to improve refolding yields through the stabilization of the protein in solution and shielding hydrophobic patches. Progress has been made in the field of high-pressure renaturation and on-column refolding. Mixing times of the denatured protein in the refolding buffer have been reduced using newly developed devices and the introduction of specific mixers. Concepts of continuous refolding have been introduced to reduce tank sizes and increase yields. Some of the patents covering refolding of proteins will soon expire or have already expired. This gives more freedom to operate. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


Grant
Agency: Cordis | Branch: H2020 | Program: MSCA-ITN-ETN | Phase: MSCA-ITN-2014-ETN | Award Amount: 4.04M | Year: 2015

Chinese hamster (CHO) ovary cells are the production host for a \50 billion /yr biopharmaceuticals market. Current CHO production platforms dates to 1980 and are based primarily on media and process optimisation with little consideration to the optimization of the cellular machinery. Fortunately, with the recent sequencing of the CHO genome, an opportunity has opened to significantly advance the CHO platform. The benefit will be advanced production flexibility and a lower production cost. This ITN graduate training programme - eCHO Systems - will blend conventional molecular, cellular, and synthetic biology with genome scale systems biology training in omics data acquisition, biological network modeling, and genome engineering in three interdisciplinary topics: 1) Acquisition of large scale omics data sets and their incorporation into genome-scale mathematical models 2) Development of genome engineering tools, enabling synthetic biology 3) Application of systems and synthetic biology and genome engineering to improve performance of CHO producers The training projects are supported by 15 industrial participants, which will participate in the research and test the results. ESR training will include intense courses focused on computational systems biology, cell biology, business and entrepreneurship. The three universities bring unique complementary skills in systems and synthetic biology, omics technologies, cytometry, and molecular cell biology which will provide depth and breadth to this training. The eCHO Systems will produce four major outputs: General knowledge to improve the productivity, quality, and efficiency of CHO platform cell lines, new systems models for CHO cells, new CHO cell line chassises generated through synthetic biology approaches, high quality education at the graduate level, and a cadre of interdisciplinary graduates poised to transform biopharmaceutical biotechnology.


Minow B.,Rentschler Biotechnologie GmbH | Tschoepe S.,Rentschler Biotechnologie GmbH | Regner A.,Rentschler Biotechnologie GmbH | Populin M.,Rentschler Biotechnologie GmbH | And 3 more authors.
Engineering in Life Sciences | Year: 2014

Process transfer is associated with a considerable risk potential. The most critical equipment aspects in upstream operations are the type and scale of bioreactors. Single-use systems have the advantage of a relatively fixed bioreactor design where only few adaptations can be made, e.g. in stirrer geometry or type of submerse aeration. Here, we describe the transfer of a Chinese hamster ovary fed-batch process in the 1000 L scale from a XDR™ to a Thermo Scientific Hyclone Single-Use Bioreactor (S.U.B.) used for GMP compliant manufacturing of biologics. The transfer method, which was based on a preceding intensive characterization of both bioreactors, aimed either to keep the oxygen mass transfer or the power input constant. The transfer strategies were evaluated theoretically based on derived empirical correlations for the mass transfer coefficients, kLaO2 and kLaCO2. An operation boundary of 10-31 W m-3 for the S.U.B. bioreactor was defined, which is an approximately 35 % higher power input compared to that in the XDR™. The transfer strategy succeeded in maintaining essential biological parameters such as cell concentration (±5%), viability (±2%), and product formation (±3%) very similar. This is, to the authors' knowledge, the first time that distinct process performance comparison in different 1000 L SUBs is published. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Minow B.,Rentschler Biotechnologie GmbH | Seidemann J.,Rentschler Biotechnologie GmbH | Tschoepe S.,Rentschler Biotechnologie GmbH | Gloeckner A.,Rentschler Biotechnologie GmbH | Neubauer P.,TU Berlin
Engineering in Life Sciences | Year: 2014

In mammalian cell culture, single-use bioreactors are widely used. Different hardware designs are available, ranging from stirred tank reactors to wave mixed and cubical shaken systems. Unlike in stainless steel systems, where standards exist, in single-use bioreactors aeration devices are often predefined by the supplier. While ring sparger systems are the gold standard in stainless steel bioreactors, not all single-use bioreactors are available with ring spargers. In this study, a comprehensive characterization of two stirred tank single-use bioreactor systems (XDR™ from Xcellerex and S.U.B. from Thermo Scientific Hyclone) was performed under GMP conditions with 200/250 L and 1000 L bioreactors. Engineering facts like mass transfer rates for oxygen kLao2 and carbon dioxide kLaCO2 as well as mixing number were evaluated. To achieve improved similarity in key engineering parameters and in consequence cell culture performance, the submerse aeration device of the S.U.B. (to date only open tube and frit) was remodeled resulting in a drilled hole sparger design. Results of the characterization showed that kLao2 in the S.U.B. was enhanced from 8.5 h-1 to 11.5 h-1 at the maximum, and the kLaCO2 was very similar between both bioreactor types. Knowledge of the characterization data as well as improved oxygen transfer rate in the S.U.B. allows for an interchangeable usage of the two different single-use bioreactors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Minow B.,Rentschler Biotechnologie GmbH | De Witt H.,Cellca GmbH | Knabben I.,Rentschler Biotechnologie GmbH
Chemie-Ingenieur-Technik | Year: 2013

A systematic and integrated use of single-use technologies was combined with a robust monoclonal antibody platform, which led to a substantial reduction of manufacturing costs, reduced timelines and increased flexibility in clinical manufacturing. A direct scale-up of a high titer monoclonal antibody-expressing CHO DG44-based cell culture platform was performed from shake flasks to a 1000 L production scale in a completely single-use manufacturing facility. The scale-up was done on the basis of calculating the specific volumetric power input which allowed a direct transfer from small culture volumes to the production scale. The timelines for process development were reduced to 3 months from the Research Cell Bank to the drug substance with highly optimized cells and appropriate culture conditions. Copyright © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.


The invention relates to methods and reagents for producing DNA vectors, in particular minicircle (MC) DNA vectors, in superhelical form. The invention further relates to highly pure preparations of circular DNA vectors, in particular MC DNA vectors.


PubMed | Rentschler Biotechnologie GmbH
Type: Journal Article | Journal: Biotechnology letters | Year: 2016

To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development.Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios.Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

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