Fabricating electrospun cellulose nanofibre adsorbents for ion-exchange chromatography

Protein separation is an integral step in biopharmaceutical manufacture with diffusion-limited packed bed chromatography remaining the default choice for industry. Rapid bind-elute separation using convective mass transfer media offers advantages in productivity by operating at high flowrates. Electrospun nanofibre adsorbents are a non-woven fibre matrix of high surface area and porosity previously investigated as a bioseparation medium. The effects of compression and bed layers, and subsequent heat treatment after electrospinning cellulose acetate nanofibres were investigated using diethylaminoethyl (DEAE) or carboxylate (COO) functionalisations. Transbed pressures were measured and compared by compression load, COO adsorbents were 30%, 70% and 90% higher than DEAE for compressions 1, 5 and 10 MPa, respectively, which was attributed to the swelling effect of hydrophilic COO groups. Dynamic binding capacities (DBCs) at 10% breakthrough were measured between 2000 and 12,000 CV/h (2 s and 0.3 s residence times) under normal binding conditions, and DBCs increased with reactant concentration from 4 to 12 mg BSA/mL for DEAE and from 10 to 21 mg lysozyme/mL for COO adsorbents. Comparing capacities of compression loads applied after electrospinning showed that the lowest load tested, 1 MPa, yielded the highest DBCs for DEAE and COO adsorbents at 20 mg BSA/mL and 27 mg lysozyme/mL, respectively. At 1 MPa, DBCs were the highest for the lowest flowrate tested but stabilised for flowrates above 2000 CV/h. For compression loads of 5 MPa and 10 MPa, adsorbents recorded lower DBCs than 1 MPa as a result of nanofibre packing and reduced surface area. Increasing the number of bed layers from 4 to 12 showed decreasing DBCs for both adsorbents. Tensile strengths were recorded to indicate the mechanical robustness of the adsorbent and be related to packing the nanofibre adsorbents in large scale configurations such as pleated cartridges. Compared with an uncompressed adsorbent, compressions of 1, 5 and 10 MPa showed increases of 30%, 110% and 110%, respectively, for both functionalisations. The data presented show that capacity and mechanical strength can be balanced through compression after electrospinning and is particular to different functionalisations. This trade-off is critical to the development of nanofibre adsorbents into different packing configurations for application and scale up in bioseparation

Nanofiber adsorbents for high productivity continuous downstream processing

An ever increasing focus is being placed on the manufacturing costs of biotherapeutics. The drive towards continuous processing offers one opportunity to address these costs through the advantages it offers. Continuous operation presents opportunities for real-time process monitoring and automated control with potential benefits including predictable product specification, reduced labour costs, and integration with other continuous processes. Specifically to chromatographic operations continuous processing presents an opportunity to use expensive media more efficiently while reducing their size and therefore cost. Here for the first time we show how a new adsorbent material (cellulosic nanofibers) having advantageous convective mass transfer properties can be combined with a high frequency simulated moving bed (SMB) design to provide superior productivity in a simple bioseparation. Electrospun polymeric nanofiber adsorbents offer an alternative ligand support surface for bioseparations. Their non-woven fiber structure with diameters in the sub-micron range creates a remarkably high surface area material that allows for rapid convective flow operations. A proof of concept study demonstrated the performance of an anion exchange nanofiber adsorbent based on criteria including flow and mass transfer properties, binding capacity, reproducibility and life-cycle performance. Binding capacities of the DEAE adsorbents were demonstrated to be 10 mg/mL, this is indeed only a fraction of what is achievable from porous bead resins but in combination with a very high flowrate, the productivity of the nanofiber system is shown to be significant. Suitable packing into a flow distribution device has allowed for reproducible bind-elute operations at flowrates of 2,400 cm/h, many times greater than those used in typical beaded systems. These characteristics make them ideal candidates for operation in continuous chromatography systems. A SMB system was developed and optimised to demonstrate the productivity of nanofiber adsorbents through rapid bind-elute cycle times of 7 s which resulted in a 15-fold increase in productivity compared with packed bed resins. Reproducible performance of BSA purification was demonstrated using a 2-component protein solution of BSA and cytochrome c. The SMB system exploits the advantageous convective mass transfer properties of nanofiber adsorbents to provide productivities much greater than those achievable with conventional chromatography media

Mid-infrared laser light nulling experiment using single-mode conductive waveguides

Aims: In the context of space interferometry missions devoted to the search of exo-Earths, this paper investigates the capabilities of new single mode conductive waveguides at providing modal filtering in an infrared and monochromatic nulling experiment; Methods: A Michelson laser interferometer with a co-axial beam combination scheme at 10.6 microns is used. After introducing a Pi phase shift using a translating mirror, dynamic and static measurements of the nulling ratio are performed in the two cases where modal filtering is implemented and suppressed. No additional active control of the wavefront errors is involved. Results: We achieve on average a statistical nulling ratio of 2.5e-4 with a 1-sigma upper limit of 6e-4, while a best null of 5.6e-5 is obtained in static mode. At the moment, the impact of external vibrations limits our ability to maintain the null to 10 to 20 seconds.; Conclusions: A positive effect of SM conductive waveguide on modal filtering has been observed in this study. Further improvement of the null should be possible with proper mechanical isolation of the setup.Comment: Accepted in A&A, 7 pages, 5 figure

Solid-Solid Interfacial Contact of Tubing Walls Drives Therapeutic Protein Aggregation During Peristaltic Pumping

Peristaltic pumping during bioprocessing can cause therapeutic protein loss and aggregation during use. Due to the complexity of this apparatus, root-cause mechanisms behind protein loss have been long sought. We have developed new methodologies isolating various peristaltic pump mechanisms to determine their effect on monomer loss. Closed-loops of peristaltic tubing were used to investigate the effects of peristaltic pump parameters on temperature and monomer loss, whilst two mechanism isolation methodologies are used to isolate occlusion and lateral expansion-relaxation of peristaltic tubing. Heat generated during peristaltic pumping can cause heat-induced monomer loss and the extent of heat gain is dependent on pump speed and tubing type. Peristaltic pump speed was inversely related to the rate of monomer loss whereby reducing speed 2.0-fold increased loss rates by 2.0- to 5.0-fold. Occlusion is a parameter that describes the amount of tubing compression during pumping. Varying this to start the contacting of inner tubing walls is a threshold that caused an immediate 20-30% additional monomer loss and turbidity increase. During occlusion, expansion-relaxation of solid-liquid interfaces and solid-solid interface contact of tubing walls can occur simultaneously. Using two mechanisms isolation methods, the latter mechanism was found to be most destructive and a function of solid-solid contact area, where increasing the contact area 2.0-fold increased monomer loss by 1.6-fold. We establish that a form of solid-solid contact mechanism whereby the contact solid interfaces disrupt adsorbed protein films is the root-cause behind monomer loss and protein aggregation during peristaltic pumping

Escherichia Coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles

Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an Escherichia coli-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs

Multivariate statistical data analysis of cell-free protein synthesis toward monitoring and control

The optimization and control of cell free protein synthesis (CFPS) presents an ongoing challenge due to the complex synergies and nonlinearities that cannot be fully explained in first principle models. This article explores the use of multivariate statistical tools for analyzing data sets collected from the CFPS of Cereulide monoclonal antibodies. During the collection of these data sets, several of the process parameters were modified to investigate their effect on the end‐point product (yield). Through the application of principal component analysis and partial least squares (PLS), important correlations in the process could be identified. For example, yield had a positive correlation with pH and NH3 and a negative correlation with CO2 and dissolved oxygen. It was also found that PLS was able to provide a long‐term prediction of product yield. The presented work illustrates that multivariate statistical techniques provide important insights that can help support the operation and control of CFPS processes

Flocculation on a chip: a novel screening approach to determine floc growth rates and select flocculating agents

Flocculation is a key purification step in cell-based processes for the food and pharmaceutical industry where the removal of cells and cellular debris is aided by adding flocculating agents. However, finding the best suited flocculating agent and optimal conditions to achieve rapid and effective flocculation is a nontrivial task. In conventional analytical systems, turbulent mixing creates a dynamic equilibrium between floc growth and breakage, constraining the determination of floc formation rates. Furthermore, these systems typically rely on end-point measurements only. We have successfully developed for the first time a microfluidic system for the study of flocculation under well controlled conditions. In our microfluidic device (μFLOC), floc sizes and growth rates were monitored in real time using high-speed imaging and computational image analysis. The on-line and in situ detection allowed quantification of floc sizes and their growth kinetics. This eliminated the issues of sample handling, sample dispersion, and end-point measurements. We demonstrated the power of this approach by quantifying the growth rates of floc formation under forty different growth conditions by varying industrially relevant flocculating agents (pDADMAC, PEI, PEG), their concentration and dosage. Growth rates between 12.2 μm s−1 for a strongly cationic flocculant (pDADMAC) and 0.6 μm s−1 for a non-ionic flocculant (PEG) were observed, demonstrating the potential to rank flocculating conditions in a quantitative way. We have therefore created a screening tool to efficiently compare flocculating agents and rapidly find the best flocculating condition, which will significantly accelerate early bioprocess development

Quantification of optical pulsed-plane-wave-shaping by chiral sculptured thin films

The durations and average speeds of ultrashort optical pulses transmitted through chiral sculptured thin films (STFs) were calculated using a finite-difference time-domain algorithm. Chiral STFs are a class of nanoengineered materials whose microstructure comprises parallel helicoidal nanowires grown normal to a substrate. The nanowires are $\sim$10-300 nm in diameter and $\sim1-10 \mu$m in length. Durations of transmitted pulses tend to increase with decreasing (free-space) wavelength of the carrier plane wave, while average speeds tend to increase with increasing wavelength. An increase in nonlinearity, as manifested by an intensity-dependent refractive index in the frequency domain, tends to increase durations of transmitted pulses and decrease average speeds. The circular Bragg phenomenon exhibited by a chiral STFs manifests itself in the frequency domain as high reflectivity for normally incident carrier plane waves whose circular polarization state is matched to the structural handedness of the film and whose wavelength falls in a range known as the Bragg regime; films of the opposite structural handedness reflect such plane waves little. This effect tends to distort the shapes of transmitted pulses with respect to the incident pulses, and such shaping can cause sharp changes in some measures of average speed with respect to carrier wavelength. A local maximum in the variation of one measure of the pulse duration with respect to wavelength is noted and attributed to the circular Bragg phenomenon. Several of these effects are explained via frequency-domain arguments. The presented results serve as a foundation for future theoretical and experimental studies of optical pulse propagation through causal, nonlinear, nonhomogeneous, and anisotropic materials.Comment: To appear in Journal of Modern Optic

Joule expansion of a pure many-body state

We derive the Joule expansion of an isolated perfect gas from the principles of quantum mechanics. Contrary to most studies of irreversible processes which consider composite systems, the gas many-body Hilbert space cannot be factorised into Hilbert spaces corresponding to interesting and ignored degrees of freedom. Moreover, the expansion of the gas into the entire accessible volume is obtained for pure states. Still, the number particle density is characterised by a chemical potential and a temperature. We discuss the special case of a boson gas below the Bose condensation temperature

Modal Filtering for Nulling Interferometry-First Single-Mode Conductive Waveguides in the Mid-Infrared

This paper presents the work achieved for the manufacturing and characterization of first single-mode waveguides to be used as modal filters for nulling interferometry in the mid-infrared range [4-20 um]. As very high dynamic range is mandatory for detection of Earth-like planets, modal filtering is one of the most stringent instrumental aspects. The hollow metallic waveguides (HMW) presented here are manufactured using micro-machining techniques. Single-mode behavior has been investigated in laboratory through a technique of polarization analysis while transmission features have been measured using flux relative comparison. The single-mode behavior have been assessed at lambda=10.6 um for rectangular waveguides with dimensions a=10 um and b<5.3 um with an accuracy of ~2.5 %. The tests have shown that a single-polarization state can be maintained in the waveguide. A comparison with results on multi-mode HMW is proposed. Excess losses of 2.4 dB (~ 58 % transmission) have been measured for a single-mode waveguide. In particular, the importance of coupling conditions into the waveguide is emphasized here. The goal of manufacturing and characterizing the first single-mode HMW for the mid-infrared has been achieved. This opens the road to the use of integrated optics for interferometry in the mentioned spectral range.Comment: 10 pages, 6 figures, accepted in A&