(107a) Structuring and Functionalization of Iron Oxide Nanoparticles for Continuous Automated in Situ Protein Purification (Invited)

Magnetic nano- and microparticles have become highly promising in diverse areas of biomedicine, including in vitro applications in diagnostics and downstream processing due to their highly selective manipulation possible by applying external magnetic fields. For these applications, homogeneous particle properties are of crucial importance, allowing a uniform response and defined chemical and physical characteristics. In addition, the surface chemistry must be tailored for the intended application to ensure stability of the particles against agglomeration in the desired medium as well as the targeted interaction with the biological system.

We have established a multi-step process for the fabrication of biofunctionalized iron oxide nanoparticles, and studied their application for the purification of recombinant model proteins [1]. The surface chemistry of the particles was optimized by modification with a tailored ligand system capable of forming stable metal complexes with histidine (His), which is a standard strategy for the purification of recombinant proteins. The successful modification of the nanoparticles resulted in colloidal stability in aqueous biological media and could be systematically varied. The ligand is synthesized by coupling (3-glycidoxypropyl) trimethoxysilane (GLYMO) and N_α,N_α-bis(carboxymethyl)-L-lysine generating a molecule with a moiety structurally similar to nitrilotriacetic acid (NTA) which is well-known for its use in chromatography because it forms coordination compounds with His-tagged proteins via metal ions such as nickel. Hence the ligand system is denominated GNTA. Following, the addition of nickel ions resulted in certain agglomeration due to coordinate bonds with the NTA-moiety, facilitating the magnetic separation because of the increased total magnetic moment. Subsequently, extensive purification experiments were performed, utilizing recombinant proteins fused to His₆-tags produced by genetically engineered Bacillus megaterium. The protein binding capacity and separation efficiency of the nanoparticles were measured ex situ in a number of consecutive cycles of cultivation and compared to a commercially available product. The separation performance was analyzed utilizing enzyme-linked immunosorbent assay (ELISA) (protein activity), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (protein quantity), gravimetric analysis, elemental analysis and dynamic light scattering. Moreover, in situ purification was studied by adding the particles directly to the cultivation broth of B. megaterium in shaking flasks. By establishing a complex cycle of application and regeneration, we could successfully utilize the nanoparticles for separation of the recombinant model proteins using handheld magnets, achieving separation efficiencies of > 80 % with purities exceeding 99.5 % regarding protein components over five consecutive cycles. However, while the functionality of the ligand system could be successfully regenerated, there was a deficiency of stability of the particle agglomerates over several cycles due to the coordinate bond characteristic of the particle interactions.

In order to increase the efficiency of the magnetic separation of the particles over consecutive cycles, the as-synthesized superparamagnetic nanoparticles were structured in a spray drying process resulting in micrometer-sized aggregates. Thereby, the overall size of the magnetically separable structures is not dependent on the stability of the agglomeration state. Interestingly, the aggregates exhibit superparamagnetic-like behavior even in the micrometer range. It is assumed that organic molecules originating from the synthesis solvent are still bound to the surface of the particles preventing the fusion of the nanoparticles on the level of the crystal structure. The saturation magnetization is increased slightly and the total magnetization rises with the added mass of the densely packed aggregate compared with the agglomerates. Since the surface chemistry of the particles is not affected by the spray drying process, the developed functionalization method could be directly applied to the aggregates. An equal number of binding groups available for protein separation can thereby be provided, independent of the size of the used magnetic particles. The functionalized spray-dried aggregates were evaluated in a semi-automated in situ purification of the recombinant model protein directly from a growing cultivation of B. megaterium using a lab-scale stirred tank reactor with an external separation loop using handheld magnets. A separation efficiency of > 80 % with a purity exceeding 97.5 % regarding protein components could be achieved. The loss of magnetic particles over consecutive cycles could be significantly decreased in comparison with agglomerated particles. The set-up of the lab-scale bioreactor was further developed from a semi-automated into an automated mode [2]. Herein, the separation, washing, elution of the product, and regeneration of the functionalized particles were controlled using LabView. With this set-up, in situ purifications of the previously examined model protein and also an antibody fragment were performed. With an incubation time of the magnetic particles of 10 min and subsequent pumping of the cultivation medium through the external loop, 55 % of the protein and 83 % of the antibody fragment were separated from the cultivation medium. The purity of both products was determined to be > 97.5 %.

The preparation of spray-dried magnetic aggregates was also evaluated regarding the economical aspects. The applied nonaqueous synthesis of the highly crystalline iron oxide nanoparticles is cost-intensive due to the used precursors and synthesis method. Substituting a fraction of the iron oxide nanoparticles with silica particles in the spray drying process resulted in SiO₂@Fe_xO_y-core-shell aggregates based on the segregation process known from literature when using suspensions with particles of different size [3]. Since the shell is comprised of the smaller iron oxide particles, the functionalization procedure can be maintained. Additionally, the saturation magnetization of the resulting aggregates can be adjusted by varying the amount of silica particles offering. The total expenditure of the aggregate preparation could be decreased since the synthesis procedure of the silica particles is comparatively facile (Stöber process). Appropriate choice of iron oxide content would still provide aggregates with sufficient magnetization for the magnetic separation. Mechanical stability of the spray-dried aggregates was evaluated in consideration of the intended use in continuous production and separation of proteins in stirred tank bioreactors concomitant with mechanical stress from the stirrer in the reactor as well as pumps and pipe bends in the peripheral equipment. The particle size was determined before and after stressing with ultrasonication and no decrease of the mean size was observed. Additionally, nanoindentation measurements were conducted and the strength of the aggregates is such that they are expected to withstand any stresses that could occur in the intended application in a bioreactor. Here, especially the stability of microbial cells can be taken as reference since they must not be damaged in order to ensure further growth and synthesis of the desired product.

In conclusion, the presented approach allows facile and highly efficient selective purification of recombinant proteins and antibody fragments and appears promising for large-scale applications.

[1] J. Gädke, L. Kleinfeldt, C. Schubert, M. Rohde, R. Biedendieck, G. Garnweitner, and R. Krull, "In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles," J Biotechnol, vol. 242, pp. 55-63, Jan 20 2017.

[2] J. Gädke, J.-W. Thies, L. Kleinfeldt, A. Kalinin, G. Starke, A. Lakowitz, R. Biedendieck, G. Garnweitner, A. Dietzel, and R. Krull, "Integrated in situ -purification of recombinant proteins from Bacillus megaterium cultivation using SPION in stirred tank reactors," Biochemical Engineering Journal, vol. 126, pp. 58-67, 2017.

[3] S. Zellmer, G. Garnweitner, T. Breinlinger, T. Kraft, and C. Schilde, "Hierarchical Structure Formation of Nanoparticulate Spray-Dried Composite Aggregates," ACS Nano, 2015/10/27 2015.