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August 01, 2009 (Vol. 29, No. 14)

Increasing Production Process Efficiency

ESETEC Seeks to Overcome Limitations of Periplasmic and Inclusion Body Production

  • Antibody Fragment Production

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    Figure 3. (A) Cell growth and product formation of an Anticalin as a function of fermentation time; (B) SDS-PAGE image of the purification of pegylated Anticalin

    In collaboration with MorphoSys, ESETEC proved to be highly efficient for the microbial production of antibody fragments (Fabs) from MorphoSys’ Human Combinatorial Antibody Library, HuCAL®. These Fabs have a binding affinity and therapeutic potential similar to full-length antibodies. They consist of two different polypeptide chains, the light chain and the heavy chain. Both chains have two intramolecular disulfide bridges.

    By using ESETEC, both chains were correctly transferred across the inner membrane into the periplasm. They were correctly processed, folded, and assembled noncovalently into hetero-dimeric Fab molecules. These Fabs were secreted into the culture broth and remained perfectly stable during fermentation. They can be efficiently isolated and purified.

    As comprehensive analysis has shown, the extracellularly secreted Fabs were fully functional and active compared to a reference Fab that had been produced by production in the periplasm. Fab yields obtained by using ESETEC were 40-fold higher than by secretion into the periplasm. Fermentation titers of more than 2 g/L were achieved without further optimization. Other Fab formats have also been successfully produced by ESETEC. Thus ESETEC is suitable for producing Fabs in high yield and quality but at lower cost than mammalian or microbial expression systems.

    ESETEC produced up to 5 g/L of an engineered protein scaffold, an Anticalin owned by Pieris. Anticalins specifically bind target molecules and are an interesting alternative to antibodies because they are smaller, do not require glycosylation, penetrate tissue more efficiently, and are highly stable.

    By using ESETEC, Anticalin yields have been improved 40–50 fold. Secreted Anticalin was efficiently isolated and purified from the culture broth. Two chromatographic steps were all that was needed to purify Anticalin, which was further processed by chemical modification with polyethylene glycol (PEGylation). After PEGylation, only one further chromatographic polishing step was required to achieve final product purity (Figure 3). A GMP-compliant process has now been developed.

    These case studies demonstrate that ESETEC enables high product yields and simplifies downstream processing compared to other established E. coli expression systems. It also saves time and costs in the production of recombinant, therapeutic proteins.

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