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Apr 1, 2012 (Vol. 32, No. 7)

Small-Scale Modeling and Predicting

  • Developing Predictive Tools

    Martin Jordan, Ph.D., a scientist in the Upstream Processing, Biotech Process Sciences division of Merck Serono, will discuss work aimed at establishing a 96-deep-well plate platform for fed-batch processes for use in cell line screening and process development.

    Dr. Jordan and colleagues transferred an entire fed-batch process that was being carried out in tubes or flasks on a shaker platform to 96-deep-well plates, keeping the physical conditions to which the cells were exposed as close as possible to the ones present in a bioreactor.

    Enabling this work was what Dr. Jordan describes as “an optimal combination of technologies available for the 96-well-plate format”: a robotic liquid-handling platform for automating processing steps such as media preparation, seeding, sampling, and dilutions; a deep-well culture system with controlled oxygen and carbon dioxide levels; and high-throughput analytical tools for tasks such as cell counting and titer assessment.

    He identifies the main bottleneck at present as the relatively slow turnaround time of the robotic liquid-handling system. For complex tests with variable doses for each well, process times are about two hours per plate.

    “For large tests run in parallel on multiple plates, protocols need careful adoption regarding time-critical steps,” he says.

    At Merck, cell line selection studies begin as soon as stable cell lines are established and ready for transfer from static to shaken plates.

    “We adapt cells for two weeks (about five passages) to the growth conditions in suspension before launching our evaluation in the generic fed-batch process,” explains Dr. Jordan. “Currently we can do this for up to 500 cell lines in parallel.”

    At every passage the researchers check each well for cell growth and adjust the dilution as needed. Throughout the process they use measurements of cell number and titer as indicators of growth, maximal cell density, viability profile, integral of viable cells (IVC), and productivity.

    Dr. Jordan reports that initial proof-of-concept studies have shown this strategy to be “an excellent screening tool, since a large number of cell lines can be early tested under specific process conditions. Only a few top candidates will finally be evaluated in a bioreactor.”

    The group has completed proof-of-concept with a limited number of cell lines.

    “As a next step we will do a full study with the maximal number of cell lines in parallel with the current cell line generation program for at least one project.”

  • Host Cell Proteins

    Click Image To Enlarge +
    Heat-map of 97 secreted proteins that demonstrated statistically significant differences in concentrations between low-titer, medium-titer, high-titer, and non-IgG producing DG44 cells. The hierarchical clustering dendrogram identified patterns of proteins that are potential biomarkers for IgG production titer. [Life Technologies]

    Late last year, at the BioProcess International conference in Long Beach, CA, a team of scientists from Life Technologies demonstrated the potential for developing a profile of secreted host cell proteins (HCPs) and using a subset of those proteins as an indicator of the level of recombinant protein being produced by the cells.

    By understanding how changes in the signature of the host cell secretome relate to product titer, it may be possible to use this proteomic strategy for evaluating clones upstream of selecting a high-producing production cell line, for media development and optimization, for assessing cell health and viability, and for process monitoring.

    The researchers described the use of quantitative mass spectrometry to measure changes in the levels of secreted HCPs in transfected cell pools that exhibited low, medium, or high levels of recombinant antibody expression.

    The cells were grown in chemically defined, protein-free media so the HCPs would be easily identifiable. The team identified distinct patterns of secreted HCPs in the cell pools that correlated to the varying degrees of antibody expression.

    They developed HCP fingerprints that comprised a subset of 50 proteins from the total of 450 HCPs identified on mass spectrometry. This subset showed the most dramatic concentration changes associated with variability in antibody production.

    Moving forward the researchers will refine the HCP signature related to protein titer and also try to develop a secreted HCP signature that correlated with the quality of recombinant protein produced. While the results presented in the poster are specific to the model studied, Peter G. Slade, Ph.D., staff scientist, molecular and cell biology R&D at Life Technologies, believes that the concept can be generalized and this strategy for nondisruptive monitoring of the secreted proteome can have multiple applications.

    “The model represents another tool available for people who want to get an indication of the general health of their culture and to understand how the protein profile links to other quality attributes of the culture,” says Stephen Gorfien, Ph.D., senior director, bioproduction R&D.

  • Parallel Processing

    Click Image To Enlarge +
    Dasgip, which was acquired by Eppendorf, develops, manufactures, and supplies parallel bioreactor systems and bioprocess control software.

    Earlier this year, Dasgip, a developer, manufacturer, and supplier of parallel bioreactor systems and accompanying bioprocess control software, was acquired by Eppendorf, a combination that Thomas Drescher, Ph.D., Dasgip’s CEO, calls “an excellent fit.”

    As a result of the acquisition, Eppendorf gained Dasgip’s expertise in the area of parallel bioprocessing, complementing the bioreactor product line it gained with the earlier acquisition of New Brunswick Scientific. Dasgip benefits from access to Eppendorf’s range of products and expertise in the areas of liquid, sample, and cell handling and to its global sales and service capabilities.

    Eppendorf now offers products for bioprocess research applications, development, and scale-up ranging from 30 mL to 3,000 liters.

    Falk Schneider, Ph.D., head of software development at Dasgip, describes a key trend driving the bioreactor market over the past 6–12 months. “We are recognizing an increasing demand for cultivation in a scale below 1 L in the market,” he says. “This demand is driven by applications in the fields of strain and cell line screening, media optimization, and process development.”

    Furthermore, he notes, “QbD methods have gained a broad awareness over the last year and are considered to be a tool to minimize the necessary amount of cultivation runs and at the same time gain the most valuable information out of them.”

    Dasgip recently introduced the DASbox mini bioreactor system. It is a controlled stirred tank bioreactor with a working volume of 60–250 mL. Drawing on Dasgip’s hardware and software design innovations for parallel bioprocessing, the DASbox comes in units of four, allowing for 32+ fold parallel systems to operate simultaneously.

    A 24-fold system consumes only six feet of lab bench space, according to Matthias Arnold, Ph.D., head of hardware development.

    “The burden of liquid cooling and heating has been completely eliminated by using peltier elements to control the temperature of bioreactors as well as condensors.”

    An overhead drive covers the full range from 30 rpm to 3,000 rpm and can serve cell culture as well as microbial applications at the same time. The DASbox also incorporates mass flow controlled gassing.


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