Cell therapy biomanufacturing: integrating biomaterial and flow-based membrane technologies for production of engineered T-cells.

Adoptive T-cell therapies (ATCTs) are increasingly important for the treatment of cancer, where patient immune cells are engineered to target and eradicate diseased cells. The biomanufacturing of ATCTs involves a series of time-intensive, lab-scale steps, including isolation, activation, genetic modification, and expansion of a patient's T-cells prior to achieving a final product. Innovative modular technologies are needed to produce cell therapies at improved scale and enhanced efficacy. In this work, well-defined, bioinspired soft materials were integrated within flow-based membrane devices for improving the activation and transduction of T cells. Hydrogel coated membranes (HCM) functionalized with cell-activating antibodies were produced as a tunable biomaterial for the activation of primary human T-cells. T-cell activation utilizing HCMs led to highly proliferative T-cells that expressed a memory phenotype. Further, transduction efficiency was improved by several fold over static conditions by using a tangential flow filtration (TFF) flow-cell, commonly used in the production of protein therapeutics, to transduce T-cells under flow. The combination of HCMs and TFF technology led to increased cell activation, proliferation, and transduction compared to current industrial biomanufacturing processes. The combined power of biomaterials with scalable flow-through transduction techniques provides future opportunities for improving the biomanufacturing of ATCTs.

Protein fouling during constant-flux virus filtration: Mechanisms and modeling.

As biomanufacturers consider the transition from batch to continuous processing, it will be necessary to re-examine the design and operating conditions for many downstream processes. For example, the integration of virus removal filtration in continuous biomanufacturing will likely require operation at low and constant filtrate flux instead of the high (constant) transmembrane pressures (TMPs) currently employed in traditional batch processing. The objective of this study was to examine the effect of low operating filtrate flux (5-100 L/m2 /h) on protein fouling during normal flow filtration of human serum Immunoglobulin G (hIgG) through the Viresolve® Pro membrane, including a direct comparison of the fouling behavior during constant-flux and constant-pressure operation. The filter capacity, defined as the volumetric throughput of hIgG solution at which the TMP increased to 30 psi, showed a distinct minimum at intermediate filtrate flux (around 20-30 L/m2 /h). The fouling data were well-described using a previously-developed mechanistic model based on sequential pore blockage and cake filtration, suitably modified for operation at constant flux. Simple analytical expressions for the pressure profiles were developed in the limits of very low and high filtrate flux, enabling rapid estimation of the filter performance and capacity. The model calculations highlight the importance of both the pressure-dependent rate of pore blockage and the compressibility of the protein cake to the fouling behavior. These results provide important insights into the overall impact of constant-flux operation on the protein fouling behavior and filter capacity during virus removal filtration using the Viresolve® Pro membrane.

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration.

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2 µm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.

Development of a transient inline spiking system for evaluating virus clearance in continuous bioprocessing-Proof of concept for virus filtration.

The development of continuous/connected bioprocesses requires new approaches for viral clearance validation, both for specific unit operations and for the overall process. In this study, we have developed a transient inline spiking system that can be used to evaluate virus clearance at distinct time points during prolonged operation of continuous bioprocesses. The proof of concept for this system was demonstrated by evaluating the viral clearance for a virus filtration step, both with and without a prefilter upstream of the virus filter. The residence time distribution was evaluated using a previously identified noninteracting fluorescent tracer, while viral clearance was evaluated from measurements of the virus titer in samples obtained downstream of the virus filter. The measured log reduction values (LRV) for ϕX174, minute virus of mice, xenotropic murine leukemia virus, and a noninfectious mock virus particle were all within 0.5 log of those obtained using a traditional batch virus challenge for both model and real-world process streams (LRV between 2.2 and 3.4 for ϕX174 using a single layer of virus filter). The results demonstrate the effectiveness of transient inline spiking to validate the virus clearance capabilities in continuous bioprocessing, an essential element for the adoption of these processes for products made using mammalian cell lines.

Cationic Covalent Organic Framework as an Ion Exchange Material for Efficient Adsorptive Separation of Biomolecules.

Although covalent organic frameworks (COFs) have earned significant interest in separation applications, the use of COFs in biomolecule separation remains unexplored. We examined the ionic COF Py-BPy2+-COF as an ion exchange material for biomolecule separation. After characterizing the properties of the synthesized COF with a variety of techniques, binding experiments with both large and small biomolecules were performed. High adsorption capacities of amino acids with different hydrophobicity and charge, as well as proteins of different isoelectric points and molecular weights, were determined in batch equilibrium experiments. Desorption experiments with mixtures of model proteins demonstrated an ability to successfully separate one protein from another with the selectivity hypothesized to be a combination of the isoelectric point, hydrophobicity, and ability to penetrate the crystalline material. Overall, the results demonstrated that Py-BPy2+-COF can be exploited as a robust crystalline anion exchange biomolecule separation material.

Impact of protein fouling on nanoparticle capture within the Viresolve® Pro and Viresolve® NFP virus removal membranes.

Virus filtration is a robust size-based technique that can provide the high level of viral clearance required for the production of mammalian-derived biotherapeutics such as monoclonal antibodies. Several studies have shown that the retention characteristics of some, but not all, virus filters can be significantly affected by membrane fouling, but there have been no direct measurements of how protein fouling might alter the location of virus capture within these membranes. The objective of this study was to directly examine the effect of protein fouling by human immunoglobulin G (IgG) on virus capture within the Viresolve® Pro and Viresolve® NFP membranes by scanning electron microscopy using different size gold nanoparticles. IgG fouling shifted the capture location of 20 nm gold nanoparticles further upstream within the Viresolve® Pro filter due to the constriction and/or blockage of the pores in the virus retentive region of the filter. In contrast, IgG fouling had no measurable effect on the capture of 20 nm nanoparticles in the Viresolve® NFP membrane, and IgG fouling had no effect on the capture of larger 40 and 100 nm nanoparticles in either membrane. These results provide important insights into how protein fouling alters the virus retention characteristics of different virus filters.

Visualizing and quantifying the nanoscale hydrophobicity and chemical distribution of surface modified polyethersulfone (PES) membranes.

Chemical modifications bring unique properties into polymeric membranes that may have enhanced filtration or separation efficiencies, antifouling, antimicrobial activity and selectivity. However, there is a lack of nanoscale characterization of the chemical additive distribution and the impacts of chemical modifiers or additives on membrane surface properties, especially those at the nanoscale. In this study, a series of industrially relevant polyethersulfone (PES) membranes modified with poly (ethylene glycol) (PEG) and polyvinylpyrrolidone (PVP) were analysed systematically. Particularly, hydrophobicity and chemical distribution were scrutinized by atomic force microscopy (AFM) and AFM coupled with infrared analysis capability (AFM-IR) for the first time that successfully resolved nanoscale structural and chemical properties of the chemically modified PES membranes. Our results indicated the heterogeneous spatial distribution of PVP and PEG based on their characteristic IR bands and the resulting hydrophobicity distribution on modified membrane surfaces at the nanoscale. Particularly, we established a linear correlation (R2 = 0.9449) between the measured adhesion force and water contact angles, which enabled the examination of local surface hydrophobicity. The PES membranes became more hydrophilic with the increasing blend of PVP and PEG. With AFM-IR, trace amounts (1-4%) of PVP could be identified sensitively on PES membranes based on their unique characteristic IR bands, which were not achieved by FTIR or IR mapping. Overall, these novel characterization approaches hold paramount importance for the design and quality control of polymer membrane modification and manufacturing.