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Mar 1, 2009 (Vol. 29, No. 5)

Grappling With Biologic Manufacturing Concerns

Complexity of Pathways for Product Development Requires Nimble Efforts

  • Improved Protein Expression

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    Avecia has a slew of strategies for maximizing titers of recombinant proteins and reducing development time and costs with the aim of building more effective manufacturing processes.

    “Our Paveway technology optimizes three elements: vectors, host strains, and fermentation protocols,” stated Andy Topping, director of early-phase development at Avecia Biologics. Topping profiled the company’s strategies for maximizing titers of recombinant proteins and reducing development time and costs with the aim of building more effective manufacturing processes.

    In order to build more efficient vectors, the company has examined the control elements in order to improve their performance. DNA looping is an important key to improved production because of its ability to foster cooperativity between binding sites on the DNA. It is generated by proteins or complexes of proteins binding to two different regions. When the appropriate operator genes are placed next to the target structural genes, a hair-pin effect is produced in which the repressor protein pulls together the two operators, and a loop of thousands of base pairs of DNA bulges out.

    So the simultaneous binding of the two sites drives protein production down to zero. Addition of the appropriate inducer releases the repressor and allows a burst of protein synthesis. Using this approach, protein production as high as 25% of total cell protein was observed and moreover could be tightly controlled with slower expression, better folding, and slow accumulation for many hours with continued cell growth.

    With respect to host strain selection, from a panel of E. coli candidate strains, those that displayed the best expression of recombinant proteins and the most robust fermentation performance at high cell densities were chosen. This left the fermentation platform as the third target for optimization. Avecia has made numerous changes including the use of preestablished cell bank protocols in which the strain history and other inputs are well known, and a scalable feed strategy predeveloped for high protein productive biomass levels.

    With all these improvements in place, the company was able to achieve high titers of protein production, in some cases higher than 14 grams per liter. However, such high levels of productivity require gargantuan quantities of resin, as much as 400 L for protein purification. So clearly the benefits of increased productivity cannot be realized unless the downstream bottleneck can be removed.

    To come to grips with this impasse, the company has developed a high-yield/high-capacity generic purification approach for antibody purification that consists of clarification of the culture supernatant, protein A affinity columns, and final polishing steps with AIEX resin (anion exchange resin) combined with CHT (ceramic hydroxyapatite).

    “We then asked, is there a comparable generic scheme for antibody fragment purification?” Topping explained.

    Since antibody fragments lack an Fc region (the binding portion to protein A), it was necessary to search alternative synthetic affinity resins. The Avecia team carried out a rapid screening program of many different resins and identified mixed mode cation exchange resins as being particularly promising. This proved to be quite satisfactory, although cleaning, resin capacity, and possible ligand leakage are all concerns.

    Topping argues that the Paveway technology achieves tight control with effective modulation of expression for a range of recombinant proteins. “We are able to progress rapidly from gene to high titer fermenters,” he stated, “and we now have a proven track record with many antibody fragments.”


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