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

Improving Cell Culture Optimization

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    Schematic diagram of the Fab domain exchange resulting in the generation of a bispecific antibody when combined with the KiH technology. Dark colors indicate heavy-chain domains. Light colors indicate light-chain domains. (A) Both arms of the intended bispecific antibody. (B) Design of the four chains of the bispecific antibody. Heavy-chain heterodimerization is achieved by use of the KiH technology. (C) Crossover of the complete VH-CH1 and VL-CL domains. (D and E) Crossover of only the VH and VL domains (D) or the CH1 and CL domains (E) within the Fab region of one half of the bispecific antibody. [Reprinted with permission of PNAS, July 5, 2011, vol 108, no 27, 11187-11192.]

    With much of the low-hanging fruit in cell culture optimization already plucked, progress today is centering on how to produce more complex molecules, improving bioproduction consistency and efficiency, and developing better predictive tools for media and processing performance.

    Work on multispecific antibodies isn’t new. They offer many compelling advantages, such as attacking signaling pathways whose redundancies often circumvent monospecific therapeutics. However, creating multispecific antibodies has been challenging.

    Speaking at Terrapin’s “Cell Culture World Congress” last month, Ingo Gorr, Ph.D., senior research scientist, cell culture research, Roche Pharmaceuticals, reviewed an approach to developing bispecific antibodies that overcomes many past problems.

    “Most anticancer drugs do not work 100 percent; there is usually some leakage,” said Dr. Gorr. “Whenever you can hit two targets in a signaling pathway your drug is likely to be more effective because you’re not allowing the cell to bypass and block the drug’s action.”

    According to Dr. Gorr, Roche’s CrossMab technology can convert existing antibodies into IgG-like bispecific antibodies. Two problems must be solved to produce the desired bispecific antibody exclusively and avoid a large mixture: 1) effective induction of heterodimerization of the two heavy chains and 2) discrimination between the two light-chain/heavy-chain interactions.

    To accomplish heterodimerization, Roche uses a technology invented by Genentech, which it calls “knobs into holes.”

    “Our approach is to alter amino acids in the C-terminal of the antibody. Within one of the heavy chains we build a knob consisting of large amino acids, and on the respective other side we build a hole with small amino acids so that we favor or force pairing of heterogeneous heavy chains,” he explained.

    Using this technique, Roche developed a bispecific IgG-like antibody for Ang-2 (angiopoietin-2) and VEGF-A. “We moved it into mouse testing where it was very efficient in preventing tumor growth,” said Dr. Gorr. Because the molecule is similar to naturally occurring IgGs, it has a similar half-life, is not prone to degradation, and is expected to exhibit reduced immunogenicity.

    “We get really high titers compared to other multispecific antibody production approaches because the cell recognizes it as an antibody. We also avoid production of many side products and end up with very few product-related impurities present. Development can be done in fast timelines.”

  • Fingerprinting Dry Media Quality

    Frustration over inconsistent dry media performance occasionally roils relations between suppliers and media users.

    Jörg von Hagen, Ph.D., head of process development/launch management at Merck Millipore, reviewed factors affecting dry media quality and urged cooperation between users and suppliers in adopting a fingerprinting technology—perhaps NIR—to provide rapid characterization of dry media quality.

    Dr. von Hagen identified “three pillars” that determine quality: the formulation, raw material quality, and production process. To demonstrate their impact Merck Millipore analyzed powder DMEM/F12, a well-characterized media with a fixed recipe, from several suppliers.

    Much of the study’s purpose, said Dr. von Hagen, was to demonstrate “it’s not just formulation that determines media performance. Particle size has to be considered as well. If you use some types of ball mill technology, you have an uncontrolled particle size reduction and end up with fairly wide range of very fine particles and some larger particles.” This in turn directly affects powder mixing and solubility—both of which affect media performance.

    Several key physical-chemical attributes were reviewed:

    • Appearance
    • pH
    • Osmolality
    • Humidity
    • Particle size
    • Cellular performance

    Wide variability was revealed in most parameters. “I wasn’t surprised,” said Dr. von Hagen. “Everybody is sourcing from different vendors or producing raw materials themselves, so you would expect differences.” More telling was that cell performance (viable cell densities) varied by as much as 50% from the fixed recipe.

    Seeking a simpler fingerprinting technology, Merck used NIR to characterize the samples—sidestepping many individual tests that were often carried out. NIR results were combined with cell performance using principal component analysis. In essence, NIR spectra “summarized” many of the various individual chemical-physical components and turned out to be a robust predictor of media performance.

    “You could not say by default a particular PCA score is a really good sign for all media. You need experience from historical batches,” said Dr. von Hagen. Given history, it’s possible to map PCA scores to good and bad batches, which could then be used to predict media performance.


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