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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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Overall comparison of the accumulated D1.3 concentrations obtained from cultivations with and without ISMS; symbols: measurements; lines: simulation (parameters from K11); theoretical simulated ISMS steps were equally distributed between 12 and 78 h.
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Figure 7: Overall comparison of the accumulated D1.3 concentrations obtained from cultivations with and without ISMS; symbols: measurements; lines: simulation (parameters from K11); theoretical simulated ISMS steps were equally distributed between 12 and 78 h.

Mentions: As can be seen from Figure 7, ISMS did not yield extra benefit for total D1.3 production since the total accumulated D1.3 concentrations with and without ISMS were equal in the best case. This result has been confirmed by simulation including theoretical multi step ISMS. As shown for extracellular protease that was degraded in biosuspension [49] such a model can be useful to optimize the number of ISMS steps before the experiment is actually performed. Neither an inhibition of D1.3 in the medium on D1.3 production itself nor losses of D1.3 degradation were observed in the extracellular medium. While the total D1.3 yield without ISMS was set Yno ISMS = 1, the yield with ISMS was YISMS = 0.91-1.1 and 0.64-0.84 for IDA and triazine beads, respectively. Elution efficiencies between 7 and 100% were received for both PVA-IDA-1 and -2 particle charges. It remains unclear whether low elution efficiencies are to be attributed to incomplete elution or loss of activity in the elution buffer although EDTA is reported to be a strong eluent which extracts the metal ions from IDA and disrupts interactions between proteins and chelating ligands [29]. Adsorption and elution efficiency of his-tagged proteins from IDA-functionalized magnetic beads can depend on the chelated metal ion: in literature, multi-subunit adsorption of a his-tagged protein onto chelated Cu2+ was reported which significantly limited elution efficiency [50]. Furthermore, the isolated target protein can potentially be structurally damaged in presence of reduced Cu+ ions which leads to reduced activity [29,50]. This implies to test other divalent metal ions such as Ni2+, Zn2+ or Co2+ which might lead to higher elution efficiencies [50]. Elution efficiencies of D1.3 from triazine-functionalized beads only reached 6%. In comparison, elution efficiencies of 35% were obtained for IgG [17], and the elution protocol needs further improvement. SDS-PAGE should be applied to control the purity of the elution samples.


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)

Overall comparison of the accumulated D1.3 concentrations obtained from cultivations with and without ISMS; symbols: measurements; lines: simulation (parameters from K11); theoretical simulated ISMS steps were equally distributed between 12 and 78 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Overall comparison of the accumulated D1.3 concentrations obtained from cultivations with and without ISMS; symbols: measurements; lines: simulation (parameters from K11); theoretical simulated ISMS steps were equally distributed between 12 and 78 h.
Mentions: As can be seen from Figure 7, ISMS did not yield extra benefit for total D1.3 production since the total accumulated D1.3 concentrations with and without ISMS were equal in the best case. This result has been confirmed by simulation including theoretical multi step ISMS. As shown for extracellular protease that was degraded in biosuspension [49] such a model can be useful to optimize the number of ISMS steps before the experiment is actually performed. Neither an inhibition of D1.3 in the medium on D1.3 production itself nor losses of D1.3 degradation were observed in the extracellular medium. While the total D1.3 yield without ISMS was set Yno ISMS = 1, the yield with ISMS was YISMS = 0.91-1.1 and 0.64-0.84 for IDA and triazine beads, respectively. Elution efficiencies between 7 and 100% were received for both PVA-IDA-1 and -2 particle charges. It remains unclear whether low elution efficiencies are to be attributed to incomplete elution or loss of activity in the elution buffer although EDTA is reported to be a strong eluent which extracts the metal ions from IDA and disrupts interactions between proteins and chelating ligands [29]. Adsorption and elution efficiency of his-tagged proteins from IDA-functionalized magnetic beads can depend on the chelated metal ion: in literature, multi-subunit adsorption of a his-tagged protein onto chelated Cu2+ was reported which significantly limited elution efficiency [50]. Furthermore, the isolated target protein can potentially be structurally damaged in presence of reduced Cu+ ions which leads to reduced activity [29,50]. This implies to test other divalent metal ions such as Ni2+, Zn2+ or Co2+ which might lead to higher elution efficiencies [50]. Elution efficiencies of D1.3 from triazine-functionalized beads only reached 6%. In comparison, elution efficiencies of 35% were obtained for IgG [17], and the elution protocol needs further improvement. SDS-PAGE should be applied to control the purity of the elution samples.

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