In situ magnetic separation of antibody fragments from Escherichia coli in complex media

BMC Biotechnology

Martin Cerff³  Alexander Scholz³  Matthias Franzreb¹  Iris L Batalha²  Ana Cecilia A Roque²  Clemens Posten³ 

[1] Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

[2] REQUIMTE, Departamento de Quimica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

[3] Institute of Life Science Engineering, Division of Bioprocess Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

关键词: Complex media;    Escherichia coli fermentation;    Extracellular protein;    Recombinant scFv antibody fragments;    Metal chelate beads;    Triazine beads;    Affinity ligands;    Protein purification;    Magnetic separation;    In situ product removal;   

Others  :  1123169
DOI  :  10.1186/1472-6750-13-44

 received in 2012-08-28, accepted in 2013-03-15,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Cerff et al.; licensee BioMed Central Ltd.

【 图 表 】

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