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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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(A) Comparison of CO2 off-gas fluxes from the bioreactor in medium (2) (upper panel) and medium (1) (lower panel); solid lines: measured data (exp.); dashed lines: simulated data (sim.); (B) determination of Cu2+ in the medium before and after ISMS; asterisks: Cu2+-concentrations in the medium before ISMS (medium control).
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Figure 6: (A) Comparison of CO2 off-gas fluxes from the bioreactor in medium (2) (upper panel) and medium (1) (lower panel); solid lines: measured data (exp.); dashed lines: simulated data (sim.); (B) determination of Cu2+ in the medium before and after ISMS; asterisks: Cu2+-concentrations in the medium before ISMS (medium control).

Mentions: Measurements of CO2 in the off-gas and Cu2+ concentrations in the medium were conducted to study the culture viability before and after ISMS. Decreased or no D1.3 production after ISMS correlates well with a decreased respiratory capacity of the cells: in K7 and K8, the CO2 fluxes in the off-gas stream dropped from 0.60 to 0.15 g/h and from 0.56 to 0.17 g/h after the initial ISMS step indicating a decreased metabolic activity. In K7, qCO2 recovered up to 0.34 g/h after 87 h of cultivation (Figure 6A). Interestingly, after 87 h of cultivation, BDM concentrations almost doubled although no significant D1.3 production was observed any more. This implies that cells were either inhibited to produce D1.3 after ISMS or the process suffered from microbial contamination. The second ISMS step in K7 did not evoke a drop of the CO2 signal at 116 h. In K11, no significant drop of the CO2 signal was observed indicating that the metabolic activity was not decreased by ISMS (Figure 6A). Overestimation of the CO2 flux by the simulation (Figure 6A) might be based on the fact that no overflow metabolism (e.g. acetate formation) was taken into account in the model [47].


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)

(A) Comparison of CO2 off-gas fluxes from the bioreactor in medium (2) (upper panel) and medium (1) (lower panel); solid lines: measured data (exp.); dashed lines: simulated data (sim.); (B) determination of Cu2+ in the medium before and after ISMS; asterisks: Cu2+-concentrations in the medium before ISMS (medium control).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: (A) Comparison of CO2 off-gas fluxes from the bioreactor in medium (2) (upper panel) and medium (1) (lower panel); solid lines: measured data (exp.); dashed lines: simulated data (sim.); (B) determination of Cu2+ in the medium before and after ISMS; asterisks: Cu2+-concentrations in the medium before ISMS (medium control).
Mentions: Measurements of CO2 in the off-gas and Cu2+ concentrations in the medium were conducted to study the culture viability before and after ISMS. Decreased or no D1.3 production after ISMS correlates well with a decreased respiratory capacity of the cells: in K7 and K8, the CO2 fluxes in the off-gas stream dropped from 0.60 to 0.15 g/h and from 0.56 to 0.17 g/h after the initial ISMS step indicating a decreased metabolic activity. In K7, qCO2 recovered up to 0.34 g/h after 87 h of cultivation (Figure 6A). Interestingly, after 87 h of cultivation, BDM concentrations almost doubled although no significant D1.3 production was observed any more. This implies that cells were either inhibited to produce D1.3 after ISMS or the process suffered from microbial contamination. The second ISMS step in K7 did not evoke a drop of the CO2 signal at 116 h. In K11, no significant drop of the CO2 signal was observed indicating that the metabolic activity was not decreased by ISMS (Figure 6A). Overestimation of the CO2 flux by the simulation (Figure 6A) might be based on the fact that no overflow metabolism (e.g. acetate formation) was taken into account in the model [47].

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