For the production of antibody-derived fragments, such as Fab fragments, E. coli expression systems are a cost-effective alternative to animal cell culture technologies. The primary recovery of the product, however, presents a challenge, since cell harvest, release of the product from the cells, and preparation of the final cell extract for initial purification result in a complex process scheme. Each processing step for the lysate preparation is potentially associated with a loss of product. The presence of high levels of nucleic acids from disruption of the bacterial cells can cause a considerable increase in viscosity of the cell extract and introduces an additional challenge for clarification and filtration steps. Nucleic acids, with their polyanionic charge characteristics, can naturally bind oppositely charged proteins, resulting in the formation of protein-nucleic acid complexes. The complexes tend to precipitate under unfavorable buffer conditions, such as acidic pH and low salt concentration. Complex formation involves host cell proteins but can also hold true for the recombinant target protein, especially for molecules exhibiting a basic isoelectric point, like Fab fragments. The use of DNA- and RNA-precipitating agents or degrading enzymes could improve these microbial processes greatly. We describe an investigation into the use of Benzonase® endonuclease during development of a primary recovery process for a recombinant Fab fragment from E. coli cells. The process has been designed to prepare a Fab-containing lysate as a suitable starting material for subsequent chromatographic purification employing cation exchange capture. The results clearly indicate that the use of Benzonase® endonuclease was essential in achieving high product yield. Precipitation loss of Fab during the primary recovery sequence was dramatically reduced. Moreover, filterability of process streams was improved more than tenfold. A cost estimation shows that the application of Benzonase® endonuclease is a viable option to render microbial processing economical.

Benzonase® endonuclease for improved primary recovery in an E. coli-based process for Fab production

251st National Meeting (2016)

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