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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 PVA-IDA-2 magnetic particles from medium (2) containing extra salts; (A) correlated BDM and glucose as well as (B) normalized D1.3 and total protein concentrations over cultivation time (measurements = exp.); D1.3 was normalized to the concentration cD1.3before the first ISMS step at ~ 75 h for better comparison [g/L]: 0.59 (K5), 0.22 (K8), 1.87 (K7); vertical dashed lines: induction by IPTG and ISMS steps; all other lines: simulated data (sim.); total protein was also simulated but not shown for better visibility.
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Figure 3: ISMS of D1.3 by means of PVA-IDA-2 magnetic particles from medium (2) containing extra salts; (A) correlated BDM and glucose as well as (B) normalized D1.3 and total protein concentrations over cultivation time (measurements = exp.); D1.3 was normalized to the concentration cD1.3before the first ISMS step at ~ 75 h for better comparison [g/L]: 0.59 (K5), 0.22 (K8), 1.87 (K7); vertical dashed lines: induction by IPTG and ISMS steps; all other lines: simulated data (sim.); total protein was also simulated but not shown for better visibility.

Mentions: Two reference cultivations K4 and K5 were conducted without and two cultivations K7 and K8 carried out with ISMS to investigate the performance of (repeated) ISMS (Table 1). Results of K4 were similar to those in K5 and are not shown. Before induction, cells were grown in medium (1) up to a bio dry mass concentration of 5–7.5 g/L at a maximum specific growth rate of μmax = 0.35-0.44 h-1 while glucose was rapidly consumed but not limiting (Figure 3A). After induction, BDM concentrations increased up to 11.5-12.8 g/L at the end of K5, K7 and K8. Offline BDM and glucose concentrations were similar in cultivations with and without ISMS (Figure 3A).


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 PVA-IDA-2 magnetic particles from medium (2) containing extra salts; (A) correlated BDM and glucose as well as (B) normalized D1.3 and total protein concentrations over cultivation time (measurements = exp.); D1.3 was normalized to the concentration cD1.3before the first ISMS step at ~ 75 h for better comparison [g/L]: 0.59 (K5), 0.22 (K8), 1.87 (K7); vertical dashed lines: induction by IPTG and ISMS steps; all other lines: simulated data (sim.); total protein was also simulated but not shown for better visibility.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: ISMS of D1.3 by means of PVA-IDA-2 magnetic particles from medium (2) containing extra salts; (A) correlated BDM and glucose as well as (B) normalized D1.3 and total protein concentrations over cultivation time (measurements = exp.); D1.3 was normalized to the concentration cD1.3before the first ISMS step at ~ 75 h for better comparison [g/L]: 0.59 (K5), 0.22 (K8), 1.87 (K7); vertical dashed lines: induction by IPTG and ISMS steps; all other lines: simulated data (sim.); total protein was also simulated but not shown for better visibility.
Mentions: Two reference cultivations K4 and K5 were conducted without and two cultivations K7 and K8 carried out with ISMS to investigate the performance of (repeated) ISMS (Table 1). Results of K4 were similar to those in K5 and are not shown. Before induction, cells were grown in medium (1) up to a bio dry mass concentration of 5–7.5 g/L at a maximum specific growth rate of μmax = 0.35-0.44 h-1 while glucose was rapidly consumed but not limiting (Figure 3A). After induction, BDM concentrations increased up to 11.5-12.8 g/L at the end of K5, K7 and K8. Offline BDM and glucose concentrations were similar in cultivations with and without ISMS (Figure 3A).

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