Antibody Capture from Corn Endosperm Extracts by Packed Bed and Expanded Bed Adsorption

Abstract

Topical treatments of chronic infections with monoclonal antibodies will require large quantities of antibodies. Because plants have been proven capable of producing multisubunit antibodies and provide for large-scale production, they are likely hosts to enable such applications. Recovery costs must also be low because of the relatively high dosages required. Hence, we have examined the purification of a human secretory antibody from corn endosperm extracts by processing alternatives of packed bed and expanded bed adsorption (EBA). Because of the limited availability of the transgenic corn host, the system was modeled by adding the antibody to extracts of nontransgenic corn endosperm. Complete clarification of a crude extract followed by packed bed adsorption provided antibody product in 75% yield with 2.3-fold purification (with antibody accounting for 24% of total protein). The small size of the packed bed, cation-exchange resin SP-Sepharose FF and the absence of a dense core (present in EBA resins) allowed for more favorable breakthrough performance compared to EBA resins evaluated. Four adsorbents specifically designed for EBA operation, with different physical properties (size and density), chemical properties (ligand), and base matrices were tested: SP-steel core resin (UpFront Chromatography), Streamline SP and Streamline DEAE (Amersham Biosciences), and CM Hyper-Z (BioSepra/Ciphergen Biosystems). Of these, the small hyperdiffuse-style resin from BioSepra had the most favorable adsorption characteristics. However, it could not be utilized with crude feeds due to severe interactions with corn endosperm solids that led to bed collapse. UpFront SP-steel core resin, because of its relatively smaller size and hence lower internal mass transfer resistance, was superior to the Streamline resins and operated successfully with application of a crude corn extract filtered to remove all solids of >44 microm. However, the EBA performance with this adsorbent provided a yield of only 61% and purification factor of 2.1 (with antibody being 22% of total protein). Process simulation showed that capital costs were roughly equal between packed and expanded bed processes, but the EBA design required four times greater operating expenditures. The use of corn endosperm as the starting tissue proved advantageous as the amount of contaminating protein was reduced approximately 80 times compared to corn germ and approximately 600 times compared to canola. Finally, three different inlet designs (mesh, glass beads, and mechanical mixing) were evaluated on the basis of their ability to produce efficient flow distribution as measured by residence time distribution analysis. All three provided adequate distribution (axial mixing was not as limiting as mass transfer to the adsorption process), while resins with different physical properties did not influence flow distribution efficiency values (i.e., Peclet number and HETP) when operated with the same inlet design.

Recovery of proteins from corn and soybean extracts by membrane adsorption

“…Interestingly, previous reports evaluating static binding of a different model protein, beta‐glucuronidase (GUS), to anion exchange resins, both as a pure solution and in the presence of canola or corn endosperm extract, showed no reduction in binding capacity. This difference from what was observed in the present study may be explained by the fact that canola extracts12, 50 and corn endosperm extracts51, 52 each have very few native acidic proteins that would have competed for adsorption. Also, GUS has a molecular mass (∼290 kDa) that is approximately two‐thirds that of urease,53 and it may have been able to find open binding sites where urease would have been occluded by steric hindrance.…”

In situ butanol recovery from Clostridium acetobutylicum fermentations by expanded bed adsorption

“…The use of flow‐through columns effectively eliminates such concerns by isolating the adsorbent external to the bioreactor, improving the modularity of the approach. As it offers optimal separation efficiency per unit mass of adsorbent, packed‐bed adsorption (PBA), which resembles traditional chromatography with tightly packed adsorbents, has been most commonly investigated to this end. However, with unclarified broths, PBA is prone to fouling by cells and ultimately clogging at the column inlet.…”

Scaled‐up manufacturing of recombinant antibodies produced by plant cells in a 200‐L orbitally‐shaken disposable bioreactor

“…EBA chromatography is an established method for the purification of recombinant proteins from unprocessed microbial (Anspach et al, ; Johansson et al, ; Lee et al, ; Xu et al, ) and mammalian cell culture broths (Batt et al, ; Beck et al, ; Blank et al, ; Ohashi et al, ). It has also been used successfully to capture a human secretory antibody from corn endosperm extracts (Menkhaus and Glatz, ). The M12 antibody was isolated from the EBA chromatography eluate by protein A chromatography and finally transferred into storage buffer by gel filtration.…”