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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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ISMS of D1.3 by means of IDA-1 beads (upper panel) and triazine beads (middle and lower panels) in different media from shaking flask cultivations.
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Figure 5: ISMS of D1.3 by means of IDA-1 beads (upper panel) and triazine beads (middle and lower panels) in different media from shaking flask cultivations.

Mentions: Simulation of BDM and glucose concentrations over cultivation time could be validated by offline measurements for all cultivations (Figures 3A, 4A). As it was observed in all cultivations, biomass growth already declined before induction. The experimentally unknown limitation was modeled as cysteine, i.e. sulfur limitation (data not shown), and exceptionally as glucose limitation in K7 after 98 hours. Cells were considered capable of producing cysteine from sulfate that was abundant in the medium of K5, K7 and K8 to fulfill the requirements of D1.3 production and biomass growth (rcys > 0 after t ≈ 40 h), but that was not abundant in K9, K10 and K11 (rcys = 0 after induction). This can be an explanation for decreased BDM and D1.3 titers in cultivations K9-K11. Degradation of D1.3 in the medium was set to zero (rP,i,deg = 0, see Degradation of the scFv D1.3 in cultivation media section). Significant differences between the D1.3 offline data and the simulation occurred after separation in K7 and K8. While D1.3 was reproduced in the simulation, no D1.3 reproduction occurred in the offline data (Figure 3B). Simulated D1.3 and total protein courses could be validated well with experimental data in K9-K11 including the ISMS step. D1.3 production rates were comparable before and after ISMS (K11) but were not elevated compared to the reference cultivations K9 and K10 (Figure 4B). Similar results were obtained for cultivations with and without ISMS in shaking flask cultivation (SK1, Figure 5 IDA-1 particles were applied in medium (3)). In the best case similar D1.3 production rates before and after ISMS were expected because no degradation of the D1.3 was observed. In the Response of the microorganisms to the ISMS steps an explanation is given why the production of D1.3 might have been limited in K7 and K8 after the ISMS steps.


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)

ISMS of D1.3 by means of IDA-1 beads (upper panel) and triazine beads (middle and lower panels) in different media from shaking flask cultivations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: ISMS of D1.3 by means of IDA-1 beads (upper panel) and triazine beads (middle and lower panels) in different media from shaking flask cultivations.
Mentions: Simulation of BDM and glucose concentrations over cultivation time could be validated by offline measurements for all cultivations (Figures 3A, 4A). As it was observed in all cultivations, biomass growth already declined before induction. The experimentally unknown limitation was modeled as cysteine, i.e. sulfur limitation (data not shown), and exceptionally as glucose limitation in K7 after 98 hours. Cells were considered capable of producing cysteine from sulfate that was abundant in the medium of K5, K7 and K8 to fulfill the requirements of D1.3 production and biomass growth (rcys > 0 after t ≈ 40 h), but that was not abundant in K9, K10 and K11 (rcys = 0 after induction). This can be an explanation for decreased BDM and D1.3 titers in cultivations K9-K11. Degradation of D1.3 in the medium was set to zero (rP,i,deg = 0, see Degradation of the scFv D1.3 in cultivation media section). Significant differences between the D1.3 offline data and the simulation occurred after separation in K7 and K8. While D1.3 was reproduced in the simulation, no D1.3 reproduction occurred in the offline data (Figure 3B). Simulated D1.3 and total protein courses could be validated well with experimental data in K9-K11 including the ISMS step. D1.3 production rates were comparable before and after ISMS (K11) but were not elevated compared to the reference cultivations K9 and K10 (Figure 4B). Similar results were obtained for cultivations with and without ISMS in shaking flask cultivation (SK1, Figure 5 IDA-1 particles were applied in medium (3)). In the best case similar D1.3 production rates before and after ISMS were expected because no degradation of the D1.3 was observed. In the Response of the microorganisms to the ISMS steps an explanation is given why the production of D1.3 might have been limited in K7 and K8 after the ISMS steps.

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