Rapid iterative design of tandem-core virus-like particles using Escherichia coli- based cell-free protein synthesis

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Single-use primary capture technology with the promise to deliver new standards for the economics, convenience and reliability of mAb bioprocessing

Product capture chromatography has been crucial in the process development and manufacture of mAb therapeutics over the past 20 years, and in particular Protein A affinity. Chromatography is a step that has had a lot of process development time attributed to getting packed beds to perform to their maximum capability and most of the optimisation stems from limitations of the inherent media and its pack form, such as mass transfer & pressure drop limitations, channeling and bead-wall support effects. The limited throughput that this media offers has prevented this unit operation from being economically accessible in a single-use format. Here we present 3 case studies of work using this a novel nanofibre adsorbent which takes the well-developed performance characteristics of chromatography utilising the same chemical base materials and process infrastructure and delivers a productivity improvement of 50-fold while maintaining product CQAs. This huge throughput advantage enables the drug manufacture to choose whether they want to reduce this unit process size such that the chromatography cartridge’s lifetime (in terms of cycles) can be exhausted over a single batch, with the aim of making in single-use operation economically feasible, or whether they what to trade that off with operating their unit operation in a significantly reduced time period. The goal of this is not just to reduce COGs associated with chromatography, but to enable new overall processing strategies to be employed giving drug manufacturers greater flexibility in their choice of operations and thereby maximising the productivity of new and existing drug manufacturing facilities. The work presented here explores the physical properties that enable this high productivity operation and discusses the resulting product characterisation and process considerations. The industrial suitability of the nanofibre technology has been tested across a 1,000x increase in scale. Initial development work focused on high-throughput screening studies on the Tecan liquid handling system (10-50μL) and lab scale (125μL-1mL nanofibre volume). Run times of less than 3 minutes allowed the impact of key process parameters on quality attributes such as host cell protein and product concentration to be quickly optimised. This work was then scaled to a pilot purification of a 50L CHO cell culture in a single batch. In this initial feasibility study, using a 10mL prototype housing unit, a productivity of 460g/L/h was achieved, with a recovery yield over 90%. A 3-log reduction in host cell proteins was also maintained over 200 cycles, and the Protein A leaching was less than 7ppm. Please click Additional Files below to see the full abstract

Depth filter material process interaction in the harvest of mammalian cells

Upstream advances have led to increased mAb titers above 5 g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilising these to remove process-related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/mL). It focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. An increase in loading of the primary-synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m2 being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary-synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems

GFP-tagging of extracellular vesicles for rapid process development

Extracellular vesicles (EVs) act as nano-scale molecular messengers owing to their capacity to shuttle functional macromolecular cargo between cells. This intrinsic ability to deliver bioactive cargo has sparked great interest in the use of EVs as novel therapeutic delivery vehicles; investments totaling over $2 billion in 2020 alone were reported for therapeutic EVs. One of the bottlenecks facing the production of EVs is the lack of rapid and high throughput analytics to aid process development. Here CHO cells have been designed and engineered to express GFP-tagged EVs via fusion to CD81. Moreover, this study highlights the importance of parent cell characterization to ensure lack of non-fused GFP for the effective use of this quantitative approach. The fluorescent nature of resulting vesicles allowed for rapid quantification of concentration and yield across the EV purification process. In this manner, the degree of product loss was deduced by mass balance analysis of ultrafiltration processing, reconciled up to 97% of initial feed mass. The use of GFP-tagging allowed for straightforward monitoring of vesicle elution from chromatography separations and detection via western blotting. Collectively, this work illustrates the utility of GFP-tagged EVs as a quantitative and accessible tool for accelerated process development

Evaluating 3D-printed bioseparation structures using multi-length scale tomography

X-ray computed tomography was applied in imaging 3D-printed gyroids used for bioseparation in order to visualize and characterize structures from the entire geometry down to individual nanopores. Methacrylate prints were fabricated with feature sizes of 500 µm, 300 µm, and 200 µm, with the material phase exhibiting a porous substructure in all cases. Two X-ray scanners achieved pixel sizes from 5 µm to 16 nm to produce digital representations of samples across multiple length scales as the basis for geometric analysis and flow simulation. At the gyroid scale, imaged samples were visually compared to the original computed-aided designs to analyze printing fidelity across all feature sizes. An individual 500 µm feature, part of the overall gyroid structure, was compared and overlaid between design and imaged volumes, identifying individual printed layers. Internal subvolumes of all feature sizes were segmented into material and void phases for permeable flow analysis. Small pieces of 3D-printed material were optimized for nanotomographic imaging at a pixel size of 63 nm, with all three gyroid samples exhibiting similar geometric characteristics when measured. An average porosity of 45% was obtained that was within the expected design range, and a tortuosity factor of 2.52 was measured. Applying a voidage network map enabled the size, location, and connectivity of pores to be identified, obtaining an average pore size of 793 nm. Using Avizo XLAB at a bulk diffusivity of 7.00 × 10⁻¹¹ m2s⁻¹ resulted in a simulated material diffusivity of 2.17 × 10⁻¹¹ m²s⁻¹ ± 0.16 × 10⁻¹¹ m2s⁻¹

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

A step closer to industrial scale manufacture of exosomes - Adaptation of clinical grade neural stem cells from 2D to 3D culture

Exosomes derived from the clinical grade neural stem cell line CTX (ReNeuron) are the basis of a new class of therapy for the treatment of degenerative disorders. Thus far we have generated CTX-derived exosomes at research scale in 2D planar cultures. Now the cell culture process needs to be scaled up in order to deliver commercially relevant quantities of exosomes that have the correct quality attributes. To meet these demands, CTX cells, which are adherent and habitually grown in a 2D static environment, must be adapted for growth in 3D agitated bioreactor systems. Please click Additional Files below to see the full abstract

Why nanofibers are a good adsorptive surface – fundamental understanding and industrial applications for mAb bioprocessing

Over the years, chromatography has proven to be a powerful and versatile technique for the purification of high value biotherapeutics. Yet, today’s preparative chromatography of biologics still, in principle, looks the same as it did several decades ago. Any improvements made have been incremental; constrained by the stationary phase format (porous beads), associated column size (bed height and pressure drop), and historical modes of operation. To address future manufacturing challenges such as high cost of goods, diversity in product portfolios, market dynamics and manufacturing flexibility, new, more radical approaches to the development of chromatography materials and towards associated modes of operations are needed. With the biotechnology industry maturing, wide spread adoption of new high tech tools/products such as high throughput analytics, automated process control, single use materials and real time data analysis is already taking place, which in turn will lead towards revisiting and a subsequent improvement of how chromatography will be operated in the future. Examples of such improvements that are already considered include high productivity operations such as simulated moving bed and rapid, or extreme, cycling regimes. Please click Additional Files below to see the full abstract

Nanofiber based lentiviral vector production

Viral vectors are an indispensable part of gene therapy clinical trials and lentiviral vectors (LVs) are becoming significant tools in the field. Unlike other retroviral vectors, they can transduce non-dividing cells thus providing for a wider range of potential applications. Current cultivation methods produce titers of 105 to 107 TU/mL of cell culture supernatant, which is not convenient for clinical trial requirements of 1011-1012 TU per patient [1], [2]. Therefore, it is necessary to concentrate the LV preparations and to remove process related impurities (e.g. serum proteins) and product related impurities, importantly including non-infective virus, as they can cause unwanted inflammation in patients. Small-scale purification can be achieved by ultracentrifugation but there are several disadvantages to this approach: the method is time consuming, there are limited scale-up possibilities, some impurities can be co-purified, and the success of the process is strongly dependent on well trained operator’s skills. Alternative methods that can provide for scalable production include tangential flow filtration (TFF) and chromatography. Currently, chromatography is dominated by porous bead stationary phases, which are optimized for purification of small proteins such as mAbs. This is not adequate for LV purification since binding sites located within particle pores are typically not accessible to macromolecular complexes such as viral vectors therefore alternative stationary phases are necessary. One such material is Puridify\u27s FibroSelect cellulose nanofibers. Due to its structural properties, this new purification platform provides high surface area and high capacity for viral vectors. High working flow rates are also possible due to excellent mass transfer properties based on convection, not diffusion that is typically seen in bead-based resins. [3]. In order to circumvent problems associated with transient plasmid transfection and the consequent removal of the plasmid material, we used a continuous producer cell line WinPac-RD [4] and HYPERFlask system for production of LV material. This vector has an RD-pro envelope protein and GFP reporter gene. The recovery through the purification process was monitored by several different methods: infectivity assay utilized GFP expression determined by flow cytometry, LV RNA genome was quantified via RT-qPCR using primers specific for GFP gene, LV particles were detected with p24 ELISA and SYBR Green I-based product-enhanced reverse transcriptase (SG-PERT) assays. By using TFF we were able to remove more than 99% of cell culture proteins, but LV recovery was less than 20%. While losses caused by diafiltration could be mitigated by adding stabilizing agents to the diafiltration buffer, the biggest loss occurred in the concentration step and the overall infectivity recovery remained low. This led us to investigate the implementation of a TFF-free nanofiber step based on ion-exchange chromatography to concentrate LV and eliminate a significant amount of impurities while maintaining high yield of a functional vector. [1] M. M. Segura, M. Mangion, B. Gaillet, and A. Garnier, “New developments in lentiviral vector design, production and purification.,” Expert Opin. Biol. Ther., vol. 13, no. November, pp. 987–1011, 2013. [2] R. R. MacGregor, “Clinical protocol. A phase 1 open-label clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496 in HIV-positive subjects,” Hum. Gene Ther., vol. 12, no. 16, pp. 2028–2029, 2001. [3] O. Hardick, S. Dods, B. Stevens, and D. G. Bracewell, “Nanofiber adsorbents for high productivity continuous downstream processing,” J. Biotechnol., vol. 213, pp. 74–82, 2015. [4] K. S. Sanber, S. B. Knight, S. L. Stephen, R. Bailey, D. Escors, J. Minshull, G. Santilli, A. J. Thrasher, M. K. Collins, and Y. Takeuchi, “Construction of stable packaging cell lines for clinical lentiviral vector production.,” Sci. Rep., vol. 5, p. 9021, 2015

Microfluidic chromatography for early stage evaluation of biopharmaceutical binding and separation conditions

Optimization of separation conditions for biopharmaceuticals requires evaluation of a large number of process variables. To miniaturize this evaluation a microfluidic column (1.5 mu L volume and 1cm height) was fabricated and packed with a typical process scale resin. The device was assessed by comparison to a protein separation at conventional laboratory scale. This was based upon measurement of the quality of packing and generation of breakthrough and elution curves. Dynamic binding capacities from the microfluidic column compared well with the laboratory scale. Microfluidic scale gradient elution separations also equated to the laboratory column three orders of magnitude larger in scale