Increasing batch-to-batch reproducibility of CHO-cell cultures using a model predictive control approach

By means of a model predictive control strategy it was possible to ensure a high batch-to-batch reproducibility in animal cell (CHO-cell) suspensions cultured for a recombinant therapeutic protein (EPO) production. The general control objective was derived by identifying an optimal specific growth rate taking productivity, protein quality and process controllability into account. This goal was approached indirectly by controlling the oxygen mass consumed by the cells which is related to specific biomass growth rate and cell concentration profile by manipulating the glutamine feed rate. Process knowledge represented by a classical model was incorporated into the model predictive control algorithm. The controller was employed in several cultivation experiments. During these cultivations, the model parameters were adapted after each sampling event to cope with changes in the process’ dynamics. The ability to predict the state variables, particularly for the oxygen consumption, led to only moderate changes in the desired optimal operational trajectories. Hence, nearly identical oxygen consumption profiles, cell and protein titers as well as sialylation patterns were obtained for all cultivation runs.     

DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells

Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo . This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.     

Prediction of potential GPI-modification sites in proprotein sequences.

Glycosylphosphatidylinositol (GPI) lipid anchoring is a common posttranslational modification known mainly from extracellular eukaryotic proteins. Attachment of the GPI moiety to the carboxyl terminus (omega-site) of the polypeptide follows after proteolytic cleavage of a C-terminal propeptide. For the first time, a new prediction technique locating potential GPI-modification sites in precursor sequences has been applied for large-scale protein sequence database searches. The composite prediction function (with separate parametrisation for metazoan and protozoan proteins) consists of terms evaluating both amino acid type preferences at sequence positions near a supposed omega-site as well as the concordance with general physical properties encoded in multi-residue correlation within the motif sequence. The latter terms are especially successful in rejecting non-appropriate sequences from consideration. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set of fully annotated sequences from the SWISS-PROT database as well as with a newly created database "big-Pi" (more than 300 GPI-motif mutations extracted from original literature sources). The accuracy of predicting the effect of mutations in the GPI sequence motif was above 83 %. Lists of potential precursor proteins which are non-annotated in SWISS-PROT and SPTrEMBL are presented on the WWW-page http://www.embl-heidelberg.de/beisenha/gpi/gpi_p rediction. html The algorithm has been implemented in the prototype software "big-Pi predictor" which may find application as a genome annotation and target selection tool.     

The InterPro Database, 2003 brings increased coverage and new features

InterPro, an integrated documentation resource of protein families, domains and functional sites, was created in 1999 as a means of amalgamating the major protein signature databases into one comprehensive resource. PROSITE, Pfam, PRINTS, ProDom, SMART and TIGRFAMs have been manually integrated and curated and are available in InterPro for text- and sequence-based searching. The results are provided in a single format that rationalises the results that would be obtained by searching the member databases individually. The latest release of InterPro contains 5629 entries describing 4280 families, 1239 domains, 95 repeats and 15 post-translational modifications. Currently, the combined signatures in InterPro cover more than 74% of all proteins in SWISS-PROT and TrEMBL, an increase of nearly 15% since the inception of InterPro. New features of the database include improved searching capabilities and enhanced graphical user interfaces for visualisation of the data. The database is available via a webserver (http://www.ebi.ac.uk/interpro) and anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro).     

Metabolites: a helping hand for pathway evolution?

The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evolution, such as pathway duplication, enzyme specialization, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty percent of enzyme superfamilies are quite variable, not only in changing reaction chemistry or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reactions. The most frequently occurring metabolites provide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topology of the metabolic network.     

A protocol for the update of references to scientific literature in biological databases

Entries in biological databases are usually linked to scientific references. To generate those links and to keep them up-to-date, database maintainers have to continuously scan the scientific literature to select references that are relevant for each single database entry. The continuous growth of both the corpus of scientific literature and the size of biological databases makes this task very hard. We present a protocol intended to assist the updating of an existing set of literature (abstract) links from a single database entry with new references. It consists of taking the set of MEDLINE neighbour references of the existing linked abstracts and evaluating their relevance according to the existing set of abstracts. To test the applicability of the algorithm, we did a simple benchmark of the system using the references associated with the entries of a protein domain database. Human experts found the references that the algorithm scored highly were more relevant to the database entry than those scored lowly, suggesting that the algorithm was useful.     

A complex prediction: three-dimensional model of the yeast exosome

We present a model of the yeast exosome based on the bacterial degradosome component polynucleotide phosphorylase (PNPase). Electron microscopy shows the exosome to resemble PNPase but with key differences likely related to the position of RNA binding domains, and to the location of domains unique to the exosome. We use various techniques to reduce the many possible models of exosome subunits based on PNPase to just one. The model suggests numerous experiments to probe exosome function, particularly with respect to subunits making direct atomic contacts and conserved, possibly functional residues within the predicted central pore of the complex.