Freiburg, Germany
Freiburg, Germany

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Roggenbuck D.,GA Generic Assays GmbH | Reinhold D.,Otto Von Guericke University of Magdeburg | Wex T.,Otto Von Guericke University of Magdeburg | Goihl A.,Otto Von Guericke University of Magdeburg | And 9 more authors.
Clinica Chimica Acta | Year: 2011

Background: Crohn's disease (CD) is an inflammatory bowel disease (IBD) characterized by reactivity against microbial and self antigens. Zymogen granule glycoprotein 2 (GP2) was identified as the major autoantigen of CD-specific pancreatic autoantibodies (PAB). Methods: Human GP2 was expressed in the Spodoptera frugiperda 9 (Sf9) cell line using the baculovirus system, purified by Ni-chelate chromatography, and used as antigen for anti-GP2 IgA and IgG assessment by enzyme-linked immunosorbent assays (ELISA). Antibodies to mannan of Saccharomyces cerevisiae (ASCA), PAB, and anti-GP2 were investigated in sera of 178 CD patients, 100 ulcerative colitis (UC) patients, and 162 blood donors (BD). Results: Anti-GP2 IgG and IgA were found in 48/72 (66.7%) and 23/72 (31.9%) PAB positive and 5/106 (4.7%) and 1/106 (0.9%) PAB negative CD patients (p < 0.0001), respectively. CD patients displayed significantly higher reactivity to GP2 than UC patients and BD (p < 0.0001), respectively. Occurrence of anti-GP2 antibodies correlated with PAB reactivity (Spearmen's rho = 0.493, p < 0.00001). There was a significant relationship between the occurrence of ASCA IgG and anti-GP2 IgG (p = 0.0307). Conclusions: Anti-GP2 IgG and IgA constitute novel CD specific autoantibodies, the quantification of which could improve the serological diagnosis of IBD. © 2010 Elsevier B.V.


Kloke A.,Kohler | Fiebach A.R.,Kohler | Zhang S.,Kohler | Drechsel L.,Kohler | And 9 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2014

Assay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing. To close this gap, we present the LabTube platform, which is based on assay specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid phase extraction. Process control is achieved by a centrifugally-actuated ballpen mechanism. To demonstrate the workflow and functionality of the LabTube platform, we show two LabTube automated sample preparation assays from laboratory routines: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yields were observed compared to manual reference runs, while LabTube automation could significantly reduce the hands-on-time to one minute per extraction. This journal is © the Partner Organisations 2014.


Ladd Effio C.,Karlsruhe Institute of Technology | Wenger L.,Karlsruhe Institute of Technology | Otes O.,Karlsruhe Institute of Technology | Oelmeier S.A.,Karlsruhe Institute of Technology | And 3 more authors.
Journal of Chromatography A | Year: 2015

The demand for vaccines against untreated diseases has enforced the research and development of virus-like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, yeast and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction (ATPE). The benefits of ATPE have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of polyethylene (PEG) salt aqueous two-phase systems with a high capacity of up to 4.1. mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of PEG, the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage ATPE on a centrifugal partition chromatography (CPC) device in 500. mL scale. While single-stage ATPE enabled a DNA clearance of 99.6%, multi-stage ATPE improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage ATPE to 69.1% (40.1% VLP recovery) for the multi-stage ATPE. An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%. © 2015 Elsevier B.V.


Richter C.,DIARECT AG | Richter C.,Karlsruhe Institute of Technology | Simon T.,DIARECT AG | Asen I.,DIARECT AG | And 2 more authors.
Protein Expression and Purification | Year: 2014

The autoantigen U1-68/70 K is the dominant diagnostic marker in Mixed Connective Tissue Disease (MCTD) that until recently could not be expressed in its full-length form (Northemann et al., 1995, [16]). Using cell-free expression screening, we successfully produced the snRNP protein U1-68/70 K in a soluble full-length form in Escherichia coli cells. The protein length and identity was determined by Western Blot and MS/MS analysis. Additionally, its reactivity in the autoimmune diagnostic was confirmed. Establishment of a cell-free expression system for this protein was important for further elucidation of protein expression properties such as the cDNA construct, expression temperature and folding properties; these parameters can now be determined in a fast and resource-conserving manner. © 2014 Elsevier Inc. All rights reserved.


Zimmermann S.,Karlsruhe Institute of Technology | Gretzinger S.,Karlsruhe Institute of Technology | Schwab M.-L.,Karlsruhe Institute of Technology | Schwab M.-L.,DIARECT AG | And 9 more authors.
Journal of Chromatography A | Year: 2016

As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research. © 2016 Elsevier B.V.


Berg A.,Karlsruhe Institute of Technology | Berg A.,Sanofi S.A. | Schuetz M.,Karlsruhe Institute of Technology | Schuetz M.,DIARECT AG | And 3 more authors.
Food and Bioproducts Processing | Year: 2014

Characterization of protein solubility in downstream processing steps is important to either prevent protein aggregation, e.g. during inclusion body refolding, hydrophobic interaction chromatography and formulation or to decrease solubility, e.g. for selective precipitation or crystallization. In general we distinguish between thermodynamic solubility at equilibrium and kinetically driven apparent solubility. In our study we used a high throughput screening method established on a liquid handling robot to rapidly assess an apparent solubility of lysozyme and its dependence on parameters such as pH, ionic strength and additive concentration. Combinatorial effects were measured in a reasonable amount of time with high data density and low material consumption. Parameter interactions were observed between solvent pH and temperature. With increasing margin of pH from the isoelectric point, the effect of temperature was more pronounced. In addition, we found an influence of ionic strength on the additive induced changes in apparent solubility for all systems. PEG 300 and Tween 20 improved lysozyme apparent solubility at high salt concentrations. For sorbitol and sucrose, two distinct regions of maximum apparent solubility were found depending on the additive concentration. While an explanation for single parameter effects was possible, e.g. for pH by correlating net charge and solubility, this became difficult with increasing number of parameters. By reducing the experimental effort, it was possible to build a solid data basis to elucidate the mechanism of lysozyme aggregation and to find industrial relevant regions of increased solubility. Our approach is thus a powerful tool not only for process optimization but also for an increased understanding of precipitation. © 2013 The Institution of Chemical Engineers.


Richter C.,DIARECT AG | Richter C.,Karlsruhe Institute of Technology | Konstantinidis K.,BadenBioTec GmbH | Asen I.,DIARECT AG | And 3 more authors.
Engineering in Life Sciences | Year: 2014

Lyme borreliosis is the most common tick-borne disease in North America and Europe. A two-test approach (an ELISA followed by immunoblots) for testing current and past infection has been adopted in most countries. However, the heterogeneity of Borrelia antigens (ags) and the semiquantitative character of the immunoblot remain a limitation. By combining a microarray system with cell-free expression, we established a procedure for the expression and subsequent printing of different Borrelia ags onto several multiwell microarray plate surfaces. We successfully expressed and partially purified 11 immunodominant ags of Lyme borreliosis from different Borrelia species in a self-generated Escherichia coli cell-free system. Using sera from patients suffering from Lyme disease and different specific mAbs, proteins could be reproducibly detected on the microarray plates. To confirm the diagnostic outcome of the new assay, a comparison to the same cell-based expressed, purified, and printed Borrelia ags was performed. In summary, this approach serves as a proof of principle for the identification of potential biomarkers and offers the possibility of multiplex protein detection for specific diseases. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


PubMed | Karlsruhe Institute of Technology, Sartorius Stedim Biotech and Diarect AG
Type: | Journal: Journal of chromatography. A | Year: 2016

Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of quality by design: High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7 mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%.


Kloke A.,Hsg Imit Institute For Mikro Und Informationstechnik | Niekrawietz S.,Hsg Imit Institute For Mikro Und Informationstechnik | Fiebach A.R.,Hsg Imit Institute For Mikro Und Informationstechnik | Bernhardt J.,Hsg Imit Institute For Mikro Und Informationstechnik | And 6 more authors.
17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 | Year: 2013

The protein purification process is often the bottleneck for the efficient production of a large number of different proteins. Automation of these procedures is often the crux of the matter and is frequently a trade-off between efficiency and cost. Using our novel disposable LabTube cartridges we demonstrate how the process of His-tagged protein purification can be automated in standard laboratory centrifuges. LabTube cartridges include prestored reagents which are sequentially applied to a Ni-NTA purification matrix by an integrated ballpen mechanism actuated by acceleration changes of the centrifuge. Fully automated runs demonstrated similar yield and purity compared to manual purifications with sample addition as the only manual handling step. Thus, the user is available for parallel tasks during 95 % of the overall process time (33 min). In contrast manual processing requires the user to be present for 18 minutes out of the 33 minute overall process time. Since LabTube automation requires no investment in a special lab automation device, this platform lowers the market entry barrier for lab automation.


Grant
Agency: European Commission | Branch: H2020 | Program: IA | Phase: FTIPilot-1-2015 | Award Amount: 2.61M | Year: 2016

The main objective of this ID-Lyme project is to push to the market a novel test, the Ixodes kit, for the early detection of Lyme Borreliosis (LB), also known as Lyme disease. LB can usually be treated using cost-effective antibiotics at an early stage of disease. Unfortunately, standard laboratory testing is often unable to give a clear diagnosis whether a patient is infected or not. This can result in the true infection remaining untreated. This is a significant healthcare concern because of the disabling effects and the much costlier and more challenging treatment of progressive disease. This project will deliver to the market the first point-of-care diagnostic test that can identify LB infections in the early stage prior to the onset of symptoms thereby improving LB management and patient health outcomes as well as reducing LB-related healthcare costs. The project will have significant impact on EU healthcare and society, as currently over 2.6 million tests are performed in the EU each year on people suspected of LB infection. With support of the Fast Track to Innovation program by Horizon2020, a dedicated consortium of in vitro diagnostic assay developers (Innatoss Laboratories), production specialists (NPK, Diarect) and clinical experts (Medical University of Vienna) will finalize the kit design, upscale production, and test the product under real-life conditions to prepare the kit for EU-wide market introduction and implementation. The Ixodes kit will result in: 1. early and effective treatment of patients; 2. quality of life of patients; and 3. large direct and indirect cost reductions on a EU-wide level.

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