Jochen Urthaler

Bio: Jochen Urthaler is an academic researcher from Boehringer Ingelheim. The author has contributed to research in topics: Alkaline lysis & Lysis. The author has an hindex of 8, co-authored 11 publications receiving 442 citations.

Improved downstream process for the production of plasmid DNA for gene therapy.

Abstract: Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.

Mass transfer characteristics of plasmids in monoliths

Abstract: The hydrodynamic properties and pore-structure of monoliths based on functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) were characterised by pulse response experiments using different probes representing a wide range of molecular mass. On a small scale, band spreading was found to be caused to the extent of more than 90% by extra-column effects. These monoliths have large channel diameters, providing a suitable chromatography adsorbent for processing of large molecules. Dynamic and static binding capacity for plasmid DNA was investigated. For our model plasmid, consisting of 4.9 kbp, a capacity of 7 mg/mL was observed in comparison to 0.3 mg/mL for a conventional medium designed for protein separation. When plasmids were loaded on the monolith a gradual increase in pressure drop was observed. The channels filled up and the cross-sectional area available for liquid flow decreased. Therefore, a higher pressure drop was observed during elution. This is caused by (i) shrinking of the channels as effect of the high salt concentration, (ii) high viscosity of the mobile phase due to high concentration of plasmids, and (iii) an increase of the hydrodynamic radius of the plasmid with salt concentration from 45 nm at 150 mM to 70 nm at 2 M NaCl, as measured by dynamic light scattering. These types of monoliths are considered to be the preferred adsorbents for plasmid separation.

Industrial Scale cGMP Purification of Pharmaceutical Grade Plasmid‐DNA

Abstract: The advances in gene therapy and genetic vaccination based on plasmid DNA result in an increasing demand of pharmaceutical grade plasmids produced under cGMP. A generic process consisting of a cell disintegration step followed by three different chromatography steps has been developed. Cell lysis is performed by an automated continuous reactor. The clarified lysate is further purified by hydrophobic interaction, anion exchange, size exclusion chromatography and by an ultra-filtration step. The produced plasmid DNA was up to 98 % in the supercoiled form. The final genomic DNA content was lower than 10 μg/mg plasmid DNA, RNA was not detectable by agarose gel electrophoresis, the protein content was lower than 1 μg/mg plasmid DNA and the endotoxin content lower than 0.1 EU/mg plasmid DNA. An overall yield of 50 % and higher in the desired final buffer could be obtained. This process is scalable and does not require animal derived materials, detergents or organic solvents, meeting current standards for therapeutic applications.

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology

Abstract: Monoliths have consolidated their position in bioseparation. More than 200 different applications have been reported in the past two decades and their advantages compared to conventional chromatography demonstrated. These include the high mass transfer efficiency due to the convective flow enabled by the macroporous character of the matrix. Recently plasmid DNA and viruses were separated with high efficiency and cryogels and monolithic superporous agarose were developed for capture of proteins from crude homogenates and separation of microorganisms or lymphocytes. Currently four companies manufacture monoliths mainly for analytical applications although monoliths with a volume of 0.8 liter are commercially available and 8 L are available as prototypes. A book entitled "Monolithic materials: preparation, properties and applications" was published in 2003 and became standard reference of the status of this area. This review focuses on the progress in monoliths that goes beyond the scope of this reference book. Less progress has been made in the field of bioconversions in spite of the fact that monolithic supports exhibit better performance than beads in enzymatic processing of macromolecules. It appears that the scientific community has not yet realized that supports for these applications are readily available. In addition, monoliths will further substantially advance bioseparations of both small and large molecules in the future.

Latest Advances in Cryogel Technology for Biomedical Applications

Abstract: There exists a technological need for advanced materials with improved properties for emerging biomedical applications. Recent developments in macroporous materials have demonstrated their applicability as indispensable tools in biomedical research. Cryogels, which are materials with a macroporous 3D structure, are produced as a result of controlled freezing during polymerization with a highly interconnected polymer network. Cryogels’ interest lies in their ability to address some of the limitations of their hydrogel analogues. In this review, hydrogel and cryogel basic concepts are discussed as a short primer for readers unfamiliar with the cryogels literature. Next, a general overview of the methods for synthesis and characterization of cryogels is provided, highlighting key concepts relevant to cryogels and explaining their unique properties. Finally an in‐depth overview of specific technologies and fields where cryogels have been applied is given. It is argued that the latest advances in cryogel technologies are able to address challenges in bioseparation, tissue engineering, and other emerging bioengineering disciplines.