Compatibility of column inlet and adsorbent designs for processing of corn endosperm extract by expanded bed adsorption

Abstract

Corn has emerged as a viable host for expression of recombinant proteins; targeted expression to the endosperm has received particular attention. The protein extracts from corn endosperm differ from those of traditional hosts in regard to the nature of residual solids and extracted matrix contaminants. Each of these differences presents reasons for considering expanded bed adsorption for product capture and new considerations for limitations of the method. In this work three inlet-flow distribution devices (mesh, glass ballotini, and localized mixing) and six adsorbents with different physical (size and density), chemical (ligand), and base matrix properties were evaluated to determine conditions compatible with processing of crude corn endosperm extract by expanded bed adsorption.Of the inlet devices evaluated, the design with localized mixing at the inlet (as produced commercially by UpFront Chromatography A/S, Copenhagen, DK) allowed solids up to 550 Am into the column without clogging for all flow rates evaluated. A mesh at the inlet with size restriction of either 50 Am or 80 Am became clogged with very small corn particles (< 44 Am). When glass ballotini was used, large particles (550 Am) passed through for high flow rates (570 cm/h), but even small (< 44 Am) particles became trapped at a lower flow rate (180 cm/h). The physical and chemical properties of the resin determined whether solids could be eluted. The denser UpFront adsorbents allowed for complete elution of larger and more concentrated corn solids than the currently available Amersham Streamline adsorbents (Amersham Biosciences, Piscataway, NJ) as a result of the former's higher flow rate for the desired 2Â expansion (570 cm/h for UpFront vs. 180 cm/h for Streamline). All corn solids < 162 Am eluted through nonderivatized UpFront resin. Larger corn solids began to accumulate due to their elevated sedimentation velocities. Feeds of < 44 Am solids at 0.45% and 2.0% dry weight successfully eluted through ion exchange adsorbents (DEAE and SP) from UpFront. However, significant accumulation occurred when the solids size increased to a feed of < 96 Am solids, thus indicating a weak interaction between corn solids and both forms of ion exchange ligands. Expanded beds operated with Streamline ion exchange adsorbents (DEAE and SP) did not allow full elution of corn solids of < 44 Am. A hyperdiffuse style EBA resin produced by Biosepra (Ciphergen Biosystems, Fremont, CA) with CM functionality showed a severe interaction with corn solids that collapsed the expanded bed and could not be eliminated with elevated flow rates or higher salt concentration. B 2004 Wiley Periodicals, Inc.

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

“…Expanded Bed Adsorption. The subset of inlet/resin combinations that proved able to process crude corn endosperm extracts (37) dictated the choices for testing antibody adsorption with spiked feed used here. Thus, expanded bed adsorption analysis was conducted with the UpFront 20 column with magnetic mixing at the inlet; 63 mL of UpFront SP‐steel core adsorbent was added to the column, resulting in a sedimented bed height of 20 cm.…”

“…It may be possible to include an inert material in the mixing zone and restrict resin to the stable zone for more efficient utilization of the adsorbent. The mesh provides the most efficient flow distribution, but as noted by Menkhaus and Glatz (37), this inlet is most limited in ability to admit endosperm solids.…”

“…This is most likely due to the larger contact time (slower flow rates), smaller IgG molecule compared to sIgA used in this study, and higher antibody concentration in the feed stream. Unfortunately, the StreamLine‐style resins, capable of 2× expansion at lower flow rates, could not be utilized with our corn extracts due to the inability to successfully handle the solids present in the feed material (37).…”

Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants

“…At the laboratory scale, the most common disruption technique for the extraction of recombinant proteins produced in green tobacco leaf tissue is manual grinding in buffer with a pestle and mortar. High levels of active recombinant protein can be released quickly in this way (Menkhaus and Glatz, 2004). However, the presence of vascular tissue in mature leaves can make grinding more difficult.…”

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“…Although EBA has been successful in its role as an integrative separation technology, the choices made for operation had to be made carefully. Because plant solids are generally larger, denser, and at a relatively elevated concentration after extraction (typically 10-30 wt %), the inlet to the EBA column is easily clogged and/or all solids cannot be completely eluted from the column or resin/ solid interactions result in bed collapse (Bai and Glatz, 2003a;Menkhaus and Glatz, 2004a). Thus, depending on the plant material being processed, the type of column inlet design chosen (i.e., size restricted net, glass beads, or mixing device), and the properties of the resin (both physical properties and ligand chemistry), the degree of pre-EBA processing required to remove larger pieces of biomass are different.…”