Simplified and more robust EBA processes by elution in expanded bed mode

Lihme, Allan; Zafirakos, Elias; Hansen, Marie Bendix; Olander, Morten

doi:10.1007/978-94-017-1519-5_9

Cited by 7 publications

(4 citation statements)

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“…However, while the different designs may not influence adsorption (provided a minimum flow distribution is achieved), the degree of axial mixing can alter the elution characteristics more profoundly, especially in expanded bed elution. Conditions for this case should be chosen to minimize axial dispersion along with reducing flow velocity (56,57).…”

Section: Evaluation Of Inlet Flow Distribution Axial Mixing Measuresmentioning

confidence: 99%

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

Menkhaus

Glatz

2008

Biotechnol Progress

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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.

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Section: Evaluation Of Inlet Flow Distribution Axial Mixing Measuresmentioning

confidence: 99%

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

Menkhaus

Glatz

2008

Biotechnol Progress

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“…Reversed flow is not an option for the FastLine column and may not be practical for the glass bead distributor (although this was not evaluated here). While flow reversal during elution has been used extensively with the mesh (Pharmacia Biotech, 1997), elution in expanded mode is nearly as efficient as elution in packed mode with reverse flow, and has many other advantages related to ease of operation (Hjorth, 1999;Lihme et al, 1999).…”

Section: Other Factorsmentioning

confidence: 99%

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

Menkhaus

Glatz²

2004

Biotech & Bioengineering

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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.

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“…High mass transfer rates with supermacroporous materials by convection even at higher flow velocities and without much rise in pressure drop would be one possibility to increase the productivity. Another possibility of elution in EB mode29, 30 and reduction of elution volume due to higher mass transfer rates could be beneficial for development of simple and robust process. These possibilities were exploited to develop purification process for lysozyme from crude CHWE on celbeads‐CM to define useful match of process productivity and product purity.…”

Section: Introductionmentioning

confidence: 99%

Characterization of superporous cellulose matrix for high‐throughput adsorptive purification of lysozyme

Kale

Lali

2011

Biotechnology Progress

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Protein purification essentially requires macroporous adsorbents matrices, which can provide high efficiency in packed bed and expanded bed (EB) even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the superpores. Rigid spherical superporous adsorbent beads with high carboxyl group density were prepared by crosslinking of cellulose. The matrix (diameter: 100-300 μm, mean pore size: 1-3 μm, pore volume: 57-59%, and bulk density: ∼1,438 kg/m3) could be used in packed bed as well as EB for purification of various biomolecules. Attempts were made to use indigenously developed rigid, superporous crosslinked cellulose adsorbent for high-throughput purification of lysozyme from chicken egg white's extract. A typical adsorption isotherm for lysozyme in crude was well correlated with the Langmuir isotherm model. Two maxima of binding capacity on celbeads bearing carboxymethyl (celbeads-CM) group for lysozyme were observed at pH 4.5 and 7.5. Uptake kinetics showed that the diffusivity of lysozyme was 100 times higher than conventional matrices. Such superporous matrix can be used for high-throughput purification of proteins from crude feedstocks and is reflected in leveling off of height equivalent to theoretical plate vs. flow curve after threshold velocity. Optimization of binding and elution conditions resulted in overall purification of lysozyme in a high yield and purity of 98.22 and 98.8%, respectively, with purification factor of 51.54 in a single step. The overall productivity (14.21 kg/m3 h) and specific activity (2.2×10(5) U/mg) were higher than that obtained with traditional particulate resins.

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Simplified and more robust EBA processes by elution in expanded bed mode

Cited by 7 publications

References 0 publications

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

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

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

Characterization of superporous cellulose matrix for high‐throughput adsorptive purification of lysozyme

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