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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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Isotherms for scFv D1.3 on (A) PVA-IDA-1 and −2 and (B) triazine-functionalized beads; explanations: 60’/60’ means 60 min incubation of the IDA-beads in 0.1 M Cu2+-solution and 60 min for adsorption in the biosuspension; while media (1) and (3) do not contain extra salts (see Microorganism and media section), extra salts were added to medium (2); data in brackets was not fitted by the Langmuir model.
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Figure 2: Isotherms for scFv D1.3 on (A) PVA-IDA-1 and −2 and (B) triazine-functionalized beads; explanations: 60’/60’ means 60 min incubation of the IDA-beads in 0.1 M Cu2+-solution and 60 min for adsorption in the biosuspension; while media (1) and (3) do not contain extra salts (see Microorganism and media section), extra salts were added to medium (2); data in brackets was not fitted by the Langmuir model.

Mentions: Adsorption of the target protein scFv D1.3 to the beads was quantified by measuring isotherms (Figure 2A and B) that could be approximated by the Langmuir adsorption model (Equation 2-1). In the experiments maximum loads of Qmax* = 0.28 g/g and 0.08 g/g were found for IDA-1 and IDA-2 beads, respectively, when medium (2) containing extra salts was used (Figure 2A). In medium (1) without extra salts, Qmax* was significantly decreased to ~ 0.004-0.1 g/g (Figure 2A). Different maximum loads might be based on different particle production processes and are not further discussed. Highest loads of IDA beads were achieved for 60 min incubation in Cu2+-solution and 60 min adsorption. For triazine beads we found Qmax* = 0.08 g/g in medium (2) with extra salts (Figure 2B). According to the low kd-values obtained from the isotherms (kd = 0.01-0.04 g/L), all beads can be categorized as affinity adsorbents [6]. The maximum obtained load for IDA-1 beads Qmax* = 0.28 g/g is very high compared to Qmax* = 0.13 g/g for green fluorescent protein [28]. Possibly, D1.3 concentrations were overestimated by the ELISA procedure in general (see Offline analytical procedures section, preparation of the calibration standard) due to incomplete elution or loss of D1.3 activity in the elution buffer (for discussion see Comparison of the total D1.3 yield in processes with and without ISMS section.). However, the proposed ELISA method is sufficient to compare the results provided in this study among each other.


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)

Isotherms for scFv D1.3 on (A) PVA-IDA-1 and −2 and (B) triazine-functionalized beads; explanations: 60’/60’ means 60 min incubation of the IDA-beads in 0.1 M Cu2+-solution and 60 min for adsorption in the biosuspension; while media (1) and (3) do not contain extra salts (see Microorganism and media section), extra salts were added to medium (2); data in brackets was not fitted by the Langmuir model.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Isotherms for scFv D1.3 on (A) PVA-IDA-1 and −2 and (B) triazine-functionalized beads; explanations: 60’/60’ means 60 min incubation of the IDA-beads in 0.1 M Cu2+-solution and 60 min for adsorption in the biosuspension; while media (1) and (3) do not contain extra salts (see Microorganism and media section), extra salts were added to medium (2); data in brackets was not fitted by the Langmuir model.
Mentions: Adsorption of the target protein scFv D1.3 to the beads was quantified by measuring isotherms (Figure 2A and B) that could be approximated by the Langmuir adsorption model (Equation 2-1). In the experiments maximum loads of Qmax* = 0.28 g/g and 0.08 g/g were found for IDA-1 and IDA-2 beads, respectively, when medium (2) containing extra salts was used (Figure 2A). In medium (1) without extra salts, Qmax* was significantly decreased to ~ 0.004-0.1 g/g (Figure 2A). Different maximum loads might be based on different particle production processes and are not further discussed. Highest loads of IDA beads were achieved for 60 min incubation in Cu2+-solution and 60 min adsorption. For triazine beads we found Qmax* = 0.08 g/g in medium (2) with extra salts (Figure 2B). According to the low kd-values obtained from the isotherms (kd = 0.01-0.04 g/L), all beads can be categorized as affinity adsorbents [6]. The maximum obtained load for IDA-1 beads Qmax* = 0.28 g/g is very high compared to Qmax* = 0.13 g/g for green fluorescent protein [28]. Possibly, D1.3 concentrations were overestimated by the ELISA procedure in general (see Offline analytical procedures section, preparation of the calibration standard) due to incomplete elution or loss of D1.3 activity in the elution buffer (for discussion see Comparison of the total D1.3 yield in processes with and without ISMS section.). However, the proposed ELISA method is sufficient to compare the results provided in this study among each other.

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