Magnetically enhanced centrifugation for continuous biopharmaceutical processing

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.Includes bibliographical references.Effective separation and purification of biopharmaceutical products from the media in which they are produced continues to be a challenging task. Such processes usually involve multiple steps and the overall product loss can be significant. As an integrative technique, high gradient magnetic separation (HGMS), together with the application of functional magnetic particles, provides many advantages over traditional techniques. However, HGMS has a number of drawbacks; and its application is limited because it is inherently a batch process and it is difficult to recycle the magnetic nanoparticles. This thesis explores the development of a new type of continuous magnetic separation process, called magnetically enhanced centrifugation (MEC), which exploits the interactions of magnetic particles with magnetic field gradients, forced convective flows and large centrifugal forces. Magnetically susceptible wires in a uniform magnetic field facilitate the capture and aggregation of magnetic particles on wires, and a centrifugal force perpendicular to the magnetic force conveys the particle sludge parallel to the wires in a continuous mode. The primary focus of this thesis is multi-scale modeling and simulation to understand the underlying physics of MEC processes. The potential of MEC as an effective unit operation for biopharmaceutical downstream processing has been demonstrated. Unlike traditional batch-mode HGMS, MEC has a great advantage in that it can be operated continuously as magnetic particles captured on wire surface are constantly removed.(cont.) A dimensionless model for simulating the trajectories of magnetic particles in combined magnetic and flow fields has been developed. The model was first applied to single wire configurations and then extended to multi-wire arrays. It was shown that modified rhombic arrays can provide high capture efficiency while maintaining low pressure drop. It is also shown that capture efficiencies based on results for clean, particle-free wires, may be seriously in error because the particle buildup that accumulates on the wire significantly distorts the flow and the magnetic fields and thus influences the particle trajectories. The dynamic buildup growth process was treated as a moving-boundary problem. Simulation results have shown that the capture efficiency decreases dramatically as particle buildup volume increases. In addition, the influence of particle chaining under magnetic dipole-dipole forces on separation efficiency has been investigated. Magnetic particles form chains as soon as they enter a background magnetic field, and are captured in the form of particle chains. The hydrodynamic force on particle chains was calculated using a 3-D CFD simulation. The capture radius calculated with considering the chaining effect is few times as great as the capture radius calculated assuming individual particles. Bench-top MEC experiments have shown that magnetic particle buildup generally comprises two layers with distinct structures: a spiky layer with all chains parallel to the magnetic field, and a densely-packed layer near the wire.(cont.) This unique structure reflects the dominance of magnetic forces near the wire and of magnetic dipole-dipole interactions at locations further from the wire. As more and more particles accumulate on the wire surface, the centrifugal force can overcome the cohesion of the layer or the adhesion of the layer to the wire, leading to movement of the buildup material. The onset of such movement can be achieved either by increasing the centrifugal force or by increasing the buildup height. Energy and force analyses have been carried out to study various scenarios of buildup movement. For monodisperse magnetic particles, four scenarios can be expected: chain-like layer collapsing down (I), rigid body movement (II), buildup breakage (III), and mixed behavior of rigid body movement and buildup breakage (IV). A set of design formulas were derived to predict buildup structure and different scenarios. Useful scenario and operating regime diagrams were obtained. A discrete element modeling (DEM) package was developed to study the dynamics and rheological behavior of highly concentrated magnetic particle systems. For monodisperse magnetic particles, simulation results confirmed the four regions of the scenario diagram as predicted by force arguments. For polydisperse magnetic particles, DEM simulations showed that the buildup exhibits solid-like behavior when centrifugal effects are small, and liquid-like behavior with a continuous velocity profile when centrifugal effects are large.(cont.) DEM simulations were able to predict the three dimensional effects, including the buildup profiles at the wire tip. Taken together, the results of this work provide a general strategy that can be used as a starting point for the design, evaluation, and optimization of magnetically enhanced processes that are suitable for biopharmaceutical downstream processing.by Fei Chen.Ph.D

Scalable and multiplexed nanoscale imaging

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.Cataloged from PDF version of thesis.Includes bibliographical references (pages 99-107).Microscopy has facilitated the discovery of many biological insights by optically magnifying small structures in cells and tissues. However, the resolution of optical microscopy is limited by the diffraction of light to ~200-300 nm, comparable or larger to the size of many subcellular structures. In this thesis, we describe a suite of tools based on a novel super-resolution microscopy approach called Expansion microscopy. Expansion microscopy (ExM) physically expands tissues so that the resolution of ordinary microscopes is increased -5 times by leveraging the swelling properties of polyelectrolyte hydrogels. Ordinary microscopes used with ExM are more accessible and faster than the specialized optical systems designed to image beyond the diffraction limit (e.g., STORM/PALM, STED, SIM), while yielding similar performance. Expanded tissues are also optically clear, allowing for unprecedented super-resolution imaging in thick tissues and facile reagent diffusion into the sample. We have since developed a variant of ExM, called protein retention ExM, in which proteins are directly anchored to the swellable gel using a commercially available cross-linking molecule. This strategy enables ExM of genetically encoded fluorescent proteins and commercial fluorescently labeled secondary antibodies. With these advancements, ExM can be carried out with purely commercial reagents and represents a simple extension of standard histological methods used to prepare samples for imaging. Furthermore, we have developed a variant of the ExM technology that enables RNA molecules to be directly linked to the ExM gel network via a small molecule linker and isotropic expansion. This technology, termed ExFISH, enables visualization of RNAs with nanoscale precision and single molecule resolution. We have demonstrated that the covalent anchoring of RNA also enables robust repeated washing and probe hybridization steps, opening the door to combinatorial multiplexing strategies. By leveraging these benefits, we have further developed in situ analysis tools which allow for highly multiplexed imaging of RNA identity and location with nanoscale precision in intact tissues. Taken together, these tools allow for spatially mapping molecular information onto cell types and tissue structures which could be invaluable for spatially complex biological processes such as brain function, cancer heterogeneity and organismal development.by Fei Chen.Ph. D