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Mar 15, 2011 (Vol. 31, No. 6)

Expression Systems Break with Tradition

Streamlined Methods Stray from Classic Hosts to Produce Higher Yields in Fewer Steps

  • Site-Specific Integration

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    Regeneron has developed a site-specific integration cell-line platform with optimized host cell, vector, and medium to maximize cell productivity from single-gene cell lines.

    Regeneron has developed a site-specific integration cell-line platform with optimized host cell, vector, and medium to maximize cell productivity from single-gene cell lines, according to James Fandl, Ph.D., vp, protein-expression sciences. Dr. Fandl explained that traditional methods of causing a cell line to produce enough protein to conduct an experiment are imprecise, and the results are generally not predictable.

    “When using random integration methods, for example, the genetic material integrates into different locations in the cell genome each time, producing different results that are neither predictable nor reproducible.” In contrast, a site-specific cell line is both predictable and reproducible because the genetic material is inserted precisely at the same spot each time. “A site-specific cell line is easy to make, reproducible, and efficient,” he said, noting that a single researcher can produce more than 20 cell lines within four to six weeks.

    Regeneron’s more targeted approach uses site-specific recombination sites—flp/frt or cre/lox, for example—inserted into a transcriptional hotspot. Regeneron has had consistent results in its decade of experience with site-specific cell-line development.

    Isolating site-specific integrants by flow cytometry further increases throughput, and bioreactor titers currently range from 1.5 to 2.5 g/L, Dr. Fandl said. “We can, therefore, screen a lot more antibodies up front, purified from what will become the production cell line. That saves a tremendous amount of time.”

  • Streamlined Production

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    Ajinomoto’s Corynex expression system harnesses the benefits of Corynebacterium to express active, correctly folded proteins directly into the medium.

    Ajinomoto’s Corynex™ expression system is a gram-positive, fast-growing soil bacterium that produces a high yield of recombinant protein secreted directly into the culture supernatant. The process is streamlined and generates fewer impurities than the traditional E. coli production system, according to the company.

    “Many experiments that hardly produced protein in many traditional methods could be efficiently secreted in the Corynex,” reported staff scientist Yoshimi Kikuchi, Ph.D. “Furthermore, a protein that has complex structure, such as many disulfide bonds, homodimer, heterodimer, and multimer structures could be secreted as correctly folded structures possessing biological activity.” Therefore, refolding is not required.

    Dr. Kikuchi said the purification process is simple. “Since there are minimal amounts of original secreted proteins in the culture supernatant of Corynebacterium glutamicum, the purity of the target protein secreted in the Corynex is very high.” Purification requires only six steps, according to Ajinomoto’s literature, whereas the production process using E. coli listed 12 steps.

    As an example, Dr. Kikuchi elaborated, human IGF-1 could be produced efficiently using either the Corynex system or E. coli as a host strain. Using Corynex, it is produced in its correctly folded form and purified in only one-step chromatography.

    “Using E. coli as the host strain, IGF-1 accumulates in the cytoplasm as an inclusion body, for which a re-folding process is required to obtain the active form. Furthermore, the re-folded IGF-1 is purified in four-step chromatography.”

    Tests, in which living MCF-7 cells were measured four days after being stimulated by human epidermal growth factor produced naturally or by the Corynex system, showed nearly identical results.

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