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In situ magnetic separation of antibody fragments from Escherichia coli in complex media.

Cerff M, Scholz A, Franzreb M, Batalha IL, Roque AC, Posten C - BMC Biotechnol. (2013)

Bottom Line: While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium.We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads.Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies ("D1.3") produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used.

Results: Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments.

Conclusions: We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps.

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Schematic view of the integrated stirred tank reactor-separator system for the in situ removal of extracellular proteins; D1.3-loaded functionalized magnetic beads were separated from the biosuspension in a 2 L bottle without internal wire matrix when the bottle was placed in the homogenous magnetic field in the gap between the poles of a U-shaped permanent magnet.
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Figure 1: Schematic view of the integrated stirred tank reactor-separator system for the in situ removal of extracellular proteins; D1.3-loaded functionalized magnetic beads were separated from the biosuspension in a 2 L bottle without internal wire matrix when the bottle was placed in the homogenous magnetic field in the gap between the poles of a U-shaped permanent magnet.

Mentions: Two batches of polyvinyl alcohol (PVA)-coated iminodiacetate (IDA)-functionalized beads were used to chelate Cu2+-ions and specifically bind His-tagged D1.3: PVA-IDA-1 (provided by Prof. Franzreb) [28] and PVA-IDA-2 (purchased from Chemagen, Germany). Cu2+-ions were chosen as ligands because in the case of IDA-chelating ligands, the ranking of affinities of different metal ions is Cu2+ > Ni2+ > Zn2+ ≥ Co2+. These divalent ions are preferably used for purification of histidine-tagged proteins [29]. Triazine-functionalized beads (ligand 22/8) were manufactured according to the literature [17]. Disinfection of the beads and the ISMS system (Figure 1) was done by incubation in 20-30% (v/v) ethanol for 1 hour. IDA-1 and IDA-2 beads were further incubated in 0.05-0.1 mol/L Cu2+ solution for 60 min. Elution of the PVA-IDA and triazine beads was performed by applying 0.1 mol/L Na2-EDTA/PBS buffer at pH 7.4 or 0.1 mol/L glycine buffer at pH 12.3 for 1 hour, respectively. Before and after each step, at least 3-4 subsequent washing steps of the beads occurred in PBS buffer at pH 7.4 (8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4∙2 H2O, 0.24 g KH2PO4). Particle treatment was performed at 22°C and 150 rpm in a 2 L bottle (reactor experiments; Multitron incubator by Infors, Switzerland) and in 50 mL Falcon tubes at 20 rpm (shaking flask experiments; bench scale nutating shaker by VWR, USA). Maximum particle concentrations were 70 g/L for disinfection and washing steps as well as 19 g/L and 56 g/L for adsorption and elution steps, respectively. Adsorption was conducted in shaking flasks (50 mL biosuspension) for 10 min, and magnetic separation performed for another 10 min with a NdFeB block permanent magnet (Br = 1.3 T on the surface; Webcraft, Switzerland) according to the procedure described by Käppler [11]. Adsorption in the closed system (Figure 1) was conducted in a 2 L bottle with 1.5 L biosuspension for at least 30 min. Solid-solid–liquid separation followed for at least 15 min by means of a U-shaped permanent magnet (Steinert, Germany; Bmax = 0.45 T in the gap between the pole shoes). In general, adsorption was conducted at 25°C and 150 rpm. After separation, the biosuspension was further cultivated as before. Particles applied in K7 were regenerated and reused in K8. All other beads were only used once (see Table 1).


In situ magnetic separation of antibody fragments from Escherichia coli in complex media.

Cerff M, Scholz A, Franzreb M, Batalha IL, Roque AC, Posten C - BMC Biotechnol. (2013)

Schematic view of the integrated stirred tank reactor-separator system for the in situ removal of extracellular proteins; D1.3-loaded functionalized magnetic beads were separated from the biosuspension in a 2 L bottle without internal wire matrix when the bottle was placed in the homogenous magnetic field in the gap between the poles of a U-shaped permanent magnet.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750846&req=5

Figure 1: Schematic view of the integrated stirred tank reactor-separator system for the in situ removal of extracellular proteins; D1.3-loaded functionalized magnetic beads were separated from the biosuspension in a 2 L bottle without internal wire matrix when the bottle was placed in the homogenous magnetic field in the gap between the poles of a U-shaped permanent magnet.
Mentions: Two batches of polyvinyl alcohol (PVA)-coated iminodiacetate (IDA)-functionalized beads were used to chelate Cu2+-ions and specifically bind His-tagged D1.3: PVA-IDA-1 (provided by Prof. Franzreb) [28] and PVA-IDA-2 (purchased from Chemagen, Germany). Cu2+-ions were chosen as ligands because in the case of IDA-chelating ligands, the ranking of affinities of different metal ions is Cu2+ > Ni2+ > Zn2+ ≥ Co2+. These divalent ions are preferably used for purification of histidine-tagged proteins [29]. Triazine-functionalized beads (ligand 22/8) were manufactured according to the literature [17]. Disinfection of the beads and the ISMS system (Figure 1) was done by incubation in 20-30% (v/v) ethanol for 1 hour. IDA-1 and IDA-2 beads were further incubated in 0.05-0.1 mol/L Cu2+ solution for 60 min. Elution of the PVA-IDA and triazine beads was performed by applying 0.1 mol/L Na2-EDTA/PBS buffer at pH 7.4 or 0.1 mol/L glycine buffer at pH 12.3 for 1 hour, respectively. Before and after each step, at least 3-4 subsequent washing steps of the beads occurred in PBS buffer at pH 7.4 (8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4∙2 H2O, 0.24 g KH2PO4). Particle treatment was performed at 22°C and 150 rpm in a 2 L bottle (reactor experiments; Multitron incubator by Infors, Switzerland) and in 50 mL Falcon tubes at 20 rpm (shaking flask experiments; bench scale nutating shaker by VWR, USA). Maximum particle concentrations were 70 g/L for disinfection and washing steps as well as 19 g/L and 56 g/L for adsorption and elution steps, respectively. Adsorption was conducted in shaking flasks (50 mL biosuspension) for 10 min, and magnetic separation performed for another 10 min with a NdFeB block permanent magnet (Br = 1.3 T on the surface; Webcraft, Switzerland) according to the procedure described by Käppler [11]. Adsorption in the closed system (Figure 1) was conducted in a 2 L bottle with 1.5 L biosuspension for at least 30 min. Solid-solid–liquid separation followed for at least 15 min by means of a U-shaped permanent magnet (Steinert, Germany; Bmax = 0.45 T in the gap between the pole shoes). In general, adsorption was conducted at 25°C and 150 rpm. After separation, the biosuspension was further cultivated as before. Particles applied in K7 were regenerated and reused in K8. All other beads were only used once (see Table 1).

Bottom Line: While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium.We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads.Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies ("D1.3") produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used.

Results: Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments.

Conclusions: We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps.

Show MeSH
Related in: MedlinePlus