Biomedical subjects

Martin Kleinschmidt

Publications and source records attributed to Martin Kleinschmidt.

6 recordsLinked to original sources

PubMed2006

Improving the batch-to-batch reproducibility in microbial cultures during recombinant protein production by guiding the process along a predefined total biomass profile.

In industry Escherichia coli is the preferred host system for the heterologous biosynthesis of therapeutic proteins that do not need posttranslational modifications. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of a therapeutic hormone is described. The strategy is to guide the process along a predefined profile of the total biomass that was derived from a given specific growth rate profile. This profile might have been built upon experience or derived from numerical process optimization. A surprisingly simple adaptive procedure correcting for deviations from the desired path was developed. In this way the batch-to-batch reproducibility can be drastically improved as compared to the process control strategies typically applied in industry. This applies not only to the biomass but, as the results clearly show, to the product titer also.

Bioreactors↗

PubMed2006

Open-loop control of the biomass concentration within the growth phase of recombinant protein production processes.

Recombinant protein production processes are typically divided into two phases. In the first one, pure cell propagation takes place, while in the second one product formation is switched on within the cells by adding an inducer. In the initial biomass formation phase, the cell density is rather low and, hence, the measurement quantities that could be used to determine the process' state depict small values and are rather severely distorted by measurement noise. Because of these measurement problems, the fermentation cannot be reliably controlled by feedback control during this first production phase; instead, the process must be controlled in an open-loop fashion. The consequence, worked out in this paper, is to design substrate feed rate profiles for the growth phase in such a way that they are robust with respect to the main disturbances observed in practice. The robustness of the biomass formation is shown to be primarily dependent on the specific growth rate adjusted in the first hours. High batch-to-batch reproducibility can be obtained with exponential feeding profiles F(t) corresponding to specific growth rates micro(set) well below the maximal specific growth rate micro(max) of the organism. The reduction in the growth rate needed to obtain a robust process behavior depends on the inaccuracies in the initial biomass concentrations. Quantitative feed rate profiles were obtained by numerical simulation and these results were validated experimentally by means of a series of cultivation runs, where a recombinant pharmaceutical protein was produced. All experimental data confirmed the assumptions made in the robust process design study.

Bacteria↗

PubMed2006

SPOCK.CI: a multireference spin-orbit configuration interaction method for large molecules.

We present SPOCK.CI, a selecting direct multireference spin-orbit configuration interaction (MRSOCI) program based on configuration state functions. It constitutes an extension of the spin-free density functional theory/multireference configuration interaction (DFT/MRCI) code by Grimme and Waletzke [J. Chem. Phys. 111, 5645 (1999)] and includes spin-orbit interaction on the same footing with electron correlation. Key features of SPOCK.CI are a fast determination of coupling coefficients between configuration state functions, the use of a nonempirical effective one-electron spin-orbit atomic mean-field Hamiltonian, the application of a resolution-of-the-identity approximation to computationally expensive spin-free four-index integrals, and the use of an efficient multiroot Davidson diagonalization scheme for the complex Hamiltonian matrix. SPOCK.CI can be run either in ab initio mode or as semiempirical procedure combined with density functional theory (DFT/MRSOCI). The application of these techniques and approximations makes it possible to compute spin-dependent properties of large molecules in ground and electronically excited states efficiently and with high confidence. Second-order properties such as phosphorescence rates are known to converge very slowly when evaluated perturbationally by sum-over-state approaches. We have investigated the performance of SPOCK.CI on these properties in three case studies on 4H-pyran-4-thione, dithiosuccinimide, and free-base porphin. In particular, we have studied the dependence of the computed phosphorescence lifetimes on various technical parameters of the MRSOCI wave function such as the size of the configuration space, selection of single excitations, diagonalization thresholds, etc. The results are compared to the outcome of extensive quasidegenerate perturbation theory (QDPT) calculations as well as experiment. In all three cases, the MRSOCI approach is found to be superior to the QDPT expansion and yields results in very good agreement with experimental findings. For molecules up to the size of free-base porphin, MRSOCI calculations can easily be run on a single-processor personal computer. Total CPU times for the evaluation of the electronic excitation spectrum and the phosphorescence lifetime of this molecule are below 40 h.

Journal Article↗

PubMed2004

Killing of target cells by redirected granzyme B in the absence of perforin.

Granzyme B (GzmB) is a potent apoptosis-inducing serine protease of cytotoxic lymphocytes. Following receptor-mediated endocytosis, GzmB is supposed to enter the cytosol through perforin-mediated membrane disruption. We investigated whether retargeting of GzmB to Lewis Y positive surface receptors could lead to perforin-independent target cell death. We coupled recombinant GzmB to the Lewis Y-binding antibody dsFv-B3. Targeting of GzmB to Lewis Y positive cells triggered cell death with similar efficacy as dsFv-B3 targeted Pseudomonas exotoxin fragment 38 (PE38). Since GzmB was only weakly inhibited by plasma proteins, GzmB-based immunoconjugates should be useful as a new class of immunotoxins with low immunogenicity utilizing programmed cell death for therapeutic purposes.

Animals↗

PubMed2003

Design of a modular immunotoxin connected by polyionic adapter peptides.

Immunotoxins are genetically engineered fusion proteins of an antibody Fv fragment and a toxin from bacteria or plants, which function as anti-cancer therapeutics. Here, we describe a new generation of immunotoxins in which both proteins do not form a single fusion protein but are coupled specifically via cysteine-containing polyionic fusion peptides. The engineered Pseudomonas exotoxin PE38 was N-terminally fused to the peptide E(8)C. In combination with the disulfide-stabilized Fv fragment of the tumor-specific antibody B3, which was extended by the peptide R(8)CP, the fusion peptides ensured a specific and covalent coupling of the Fv fragment and the toxin. The resulting immunotoxin was as active and as specific as an immunotoxin consisting of a fusion protein of the same antibody fragment connected to the toxin.

ADP Ribose Transferases↗

PubMed2002

Spin-orbit coupling of DFT/MRCI wavefunctions: method, test calculations, and application to thiophene.

During the past decade the one-center mean-field approximation has proven to be a very appropriate framework for the accurate description of spin-orbit effects at the correlated all-electron level. Here, a new efficient code, SPOCK, is introduced that calculates spin-orbit matrix elements in the one-center mean-field approximation for multireference CI wave functions. For the first time, the computation of spin-dependent interactions within a Kohn-Sham orbital based CI (DFT/MRCI) scheme1 is made possible. The latter approach is suitable for large scale systems with up to 100-200 valence electrons. Test calculations are performed on well-known diatomic molecules and the thiocarbonyl pyranthione. Spin-orbit matrix elements show good agreement with their Hartree-Fock orbital based counterparts but are obtained at considerably lower expense, thus demonstrating the power of the method. As an application singlet-triplet couplings in thiophene are investigated that are important for the photophysics and photochemistry. Spin-orbit matrix elements between all pi --> pi* excited states are found to be small. Considerably larger spin-orbit matrix elements are observed only for cases in which pi --> sigma* excited configurations are involved.

Journal Article↗