Purification of a hepatitis C vaccine candidate: Comparison between multi- column chromatographic processes operated in positive and negative mode

Given the increasing efficiencies in bioreaction and growing interest on complex biopharmaceutical products such as virus-like particles (VLPs), downstream processing (DSP) is becoming ever more relevant. Therefore, the biopharmaceutical industry is looking for alternative downstream strategies capable of improving purification yields whilst improving product quality and lowering costs. One of most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. We report on the development and comparison of two types of multi-column chromatographic systems aimed at the purification of Hepatitis C VLPs, produced using insect cell-based expression with recombinant baculovirus. The first process described herein is based on direct product capture using an anion exchange chromatographic media and subsequent elution with the modulation of ionic strength. By using a multi-column approach, one is able to overcome the limits of dynamic binding capacity characteristic of single-column batch processes, thus increasing the media capacity utilization. The second process reported is based on negative chromatographic purification. In this approach elution conditions are such that impurities should adsorb on the chromatographic media whereas the product of interest flows through the column. Both process approaches are subjected to a temporal arrangement of operations steps suchlike column equilibration, product application, production and regeneration. Volumetric productivity thus depends not only on the optimal scheduling of the referred steps, but also upon factors such as media capacity for the product and related impurities, operational flow-rates, and mechanical limitations of the systems used. The proposed analysis compares volumetric productivity, resin capacity utilization, equipment footprint and skid complexity for both purification strategies. Also we will demonstrate that the optimal design is a balance between the manufacturing scale, complexity and imposed product quality requirements

Continuous purification of hepatitis C virus-like particles by multi-column chromatography

Novel biopharmaceutical products, such as virus-like particles (VLPs) and viral vectors, constitute a challenging task for downstream processing (DSP). Recoveries achieved to reach required purities are significantly inferior compared to more common antibody and other recombinant processes. Therefore, the biopharmaceutical industry is looking for alternative downstream strategies capable of improving purification yields and decreasing cost while maintaining product quality. One of many possible improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. A single-column batch chromatographic operation used for capture is limited by the dynamic binding capacity (DBC) of the target product. For high-value products, chromatographic columns are normally loaded to less than 1% of DBC underutilizing the resin\u27s capacity. Increasing capacity utilization leads to significant resin cost savings, particularly relevant in the case of capture with expensive affinity materials. Multi-column processes have been shown to improve process efficiency and economics, providing a powerful and flexible alternative to conventional batch chromatography. In fact, a simple serial connection of two chromatographic columns, where the effluent of the first column of the train is directed to the inlet of the second column, allows that in a loading step the breakthrough of the first column is captured on the second bed, thus avoiding product loss. After saturation, the first column can be subjected to the normal processing steps of a batch chromatographic operation while loading is resumed in the adjacent bed. Moreover, this simple setup modification allows not only to extend the utilization of the resin’s capacity, overcoming the aforementioned issues, but also to benefit from the counter-current flow between the mobile and the stationary phases, which optimizes the driving force for mass transfer throughout the overall trajectory of the two phases. We report the development of a continuous chromatographic process for the purification of Hepatitis C VLPs (HCV-VLPs), produced using insect cell-based expression with recombinant baculovirus. A library of novel anion exchange resins with different ligand densities was evaluated for improved binding and release of the target product and impurity clearance in batch operation. A model-based approach for a smooth transition from a single-column batch process to a continuous multi-column operation is demonstrated and the scheduling of periodic events of the process cycle is analyzed. The contribution of column overloading, counter current operation and faster flow rates to recovery improvements compared to batch is discussed. Ultimately, both purification strategies, batch and continuous, are compared not only in terms of volumetric productivity, resin capacity utilization and footprint reduction, but also to indicate whether actual performance can be improved by continuous operation

Improving downstream processing of enveloped virus-like particles with multi-column chromatography

The interest in continuous downstream purification processes is rapidly growing as industry pursues the establishment of continuous manufacturing. Continuous multi-column chromatography is therefore looked as an enabling technology, capable of improving purification yields whilst improving product quality and lowering costs. We report on the development and comparison of two types of multi-column chromatographic systems aimed at the purification of enveloped VLPs, produced using insect cell-based expression with recombinant baculovirus. By subjecting an array of chromatographic devices to a temporal sequence of operations steps, suchlike column equilibration, product application, production and regeneration, one is able to overcome the limits of dynamic binding capacity characteristic of single-column batch processes. This will enable the increase of volumetric productivity, column capacity utilization and subsequently a decrease on processing costs. The first process described herein is based on direct product capture using an anion exchange chromatographic media and subsequent elution with the modulation of ionic strength. The second process reported is based on negative chromatographic purification. In this approach, elution conditions are such that impurities should adsorb on the chromatographic media whereas the product of interest flows through the column. The proposed strategies will be compared in terms of their volumetric productivity, resin capacity utilization, equipment footprint and skid complexity. We will also demonstrate that the optimal design is not only a balance between the manufacturing scale, complexity and imposed product quality requirements, but depends also upon factors such as media capacity for the product and related impurities, operational flow-rates, and mechanical limitations of the systems used

Rapid, cost-effective and scalable gmp-compliant simian adenovirus-vectored vaccine production for early-phase clinical trials using entirely disposable product-contact components

The Jenner Institute, University of Oxford, develops and produces a range of vaccines against emerging threats (such as Zika) and current global health challenges (including malaria, HIV and rabies). The Jenner Clinical Biomanufacturing Facility (CBF) manufactures multiple simian adenovirus-vectored vaccines for early phase clinical trials each year. Hitherto we have used shake flasks for upstream production and caesium chloride gradient ultracentrifugation for downstream purification. This process is robust and simple but also slow, human resource intensive and lacks scalability. Here we report the development of a novel process using a 2 x 3L single-use stirred tank bioreactor system (MilliporeSigma Mobius®), coupled to a tangential flow filtration (TFF) and anion exchange chromatography (AEX)-based downstream process. The process also includes particle lysis and nucleic acid digestion inside the bioreactor, as well as clarification of cells and debris using depth filters. As our test case, we used a novel simian adenovirus-vectored rabies vaccine (ChAdOx2 RabG), which we will manufacture to GMP standards in the coming year. Each process run yields \u3e5x1013 ChAdOx2 RabG virus particles (approximately 1000 human doses), with residual host cell DNA, host cell protein and nuclease levels suitable for clinical trial use. While similar processes have been previously reported for adenovirus manufacture, we will report a number of points of novelty. Firstly, we use single-use disposable product-contact components from beginning to end, greatly simplifying small-scale GMP manufacturing of multiple products. Secondly, we will report results of comparative testing with a range of modern ion exchange media (including resins, membrane adsorbers, monoliths and functionalized hydrogel formats). Thirdly, we will report the development and validation of novel quality control methods suitable for this process. The resulting process will allow the CBF to increase production yield and produce more vaccines that transfer more easily to larger facilities

Evaluation of single-use bioreactors for the production of a Hepatitis C vaccine candidate

During the last decade, the usage of single-use bioreactors has been increasing in the biopharmaceutical industry. This technology offers some appealing advantages over their conventional counterparts made of glass or stainless steel, such as operational flexibility, faster batch-to-batch turnaround times and the reduction of clean-up and validation characteristic of single-use materials. The present work examines a stirred single-use bioreactor for its suitability for the production of a Hepatitis C Virus-Like Particles (VLPs) vaccine candidate using the baculovirus expression system with Sf9 cells. In this sense a 2L glass stirred tank and a Mobius® 3L Bioreactor are compared in terms of viable cell concentration, viability percentage, growth kinetics, stability, and VLPs production, showing that comparable results can be obtained with a simple matching of hydrodynamic working parameters between the two systems. Moreover, we report on the successful scale-up of this disposable alternative from a 3L to a 50L scale, demonstrating the potential, and ease-of-use of this technology for the production of complex biopharmaceutical products

Production and purification of influenza virus like particles using single-use technologies

There is an increasing trend in the biopharmaceutical market towards the implementation of single-use technologies (SUT) in bioprocesses. These technologies address the needs of bio therapeutic development and manufacturing with appealing advantages over the conventional systems. SUT can be operated at lower costs, eliminating the need for cleaning and regeneration of components, improving process-to-process turnaround, being more flexible enabling modular facilities, and easy to scale up. This work describes the up and downstream processing of Influenza Virus-like particles (VLPs), produced using the baculovirus expression system with High Five cells. The single-use Mobious ®Bioreactor is compared with a glass stirred tank in terms of growth kinetics, cell viability, stability and VLPs production, showing comparable results. The use of this single-use bioreactor was already reported by our group for the successful production of a hepatitis C VLP vaccine candidate. Moreover, we report on the development of a single-use platform process for purification of Influenza VLPs. We have undertaken an effort to replace chromatographic steps from our platform, with the ultimate goal of an all filtration purification process. The proposed process employs either normal or tangential flow filtration for the clarification stage, followed by a cascade of ultrafiltration steps with different pore sizes and a sterile filtration step to achieve the needed concentration and purity specifications. Efforts to clear nucleic acid without the use of an endonuclease digestion step and the impact on the downstream unitary operations will also be described. By optimizing the filtration mode of operation we were able to achieve product recoveries of 80%. Globally, we have about 1.8 log reduction value (LRV) of DNA and total protein removal and a baculovirus’ LRV of 4. Overall, using SUT across all biomanufacturing operations we are able to speed up the process, to improve the scale-up and to reduce costs due to the removal of chromatographic and cleaning and validation steps

Enveloped virus-like particles purification using an all-filtration technology platform

Virus-like particles (VLPs) have become widely used as vaccine candidates because of their versatility, immunogenicity and safety profile. The diversity of surface epitopes contributes, however, to a variability in downstream purification that could ultimately affect manufacturability. For baculovirus expression systems in particular, the similarity between residual baculovirus and VLP particles causes significant problems. For that purpose, we have undertaken an effort to develop platform processes for purification of VLPs. Our initial approach focus on size exclusion as the key mechanism of separation, with the ultimate goal of an all filtration purification process, inserted in the “anything but chromatography” concept. The first step was to evaluate a legacy purification that was not robust or efficient and replace the ion exchange chromatography step with size exclusion chromatography (SEC). Performance of the SEC step will be described and the shortcomings of such a method for a scaled up, GMP process will be discussed. The proposed all-filtration process employs either normal or tangential flow filtration for the clarification stage, followed by a cascade of ultrafiltration steps with different pore sizes and a sterile filtration step to achieve the needed concentration and purity specifications. Efforts to clear nucleic acid without the use of an endonuclease digestion step and the impact on the downstream unitary operations will also be described. By optimizing the filtration mode of operation we were able to achieve product recoveries above 85%. Globally, we have about 90% of DNA and total protein removal and a baculovirus’ log reduction value of 6. Using this all-filtration platform we are able to speed up the process, to improve the scale-up and to reduce costs due to the removal of chromatographic steps. To show the potential for a universal, platform process, two cell systems producing two different VLPs were studied and preliminary results will be presented

Evaluation and scale-up of single-use bioreactors for the production and harvesting of a hepatitis C vaccine candidate

The present work examines the suitability of single-use bioreactors for production of a Hepatitis C Virus-Like Particle (VLP) vaccine candidate using the baculovirus expression system with Sf9 cells. It can be shown that a Mobius® 3L bioreactor results in viable cell concentration, viability, growth kinetics, stability and VLP production that are comparable to standard glass bioreactors. A simple translation of hydrodynamic working parameters between the two systems is adequate to match performance. Furthermore, we report on the successful scale-up of this disposable alternative from a 3L to a 50L scale using minimal optimization. These results demonstrate the potential and ease of use of this technology for the production of complex biopharmaceutical products. Using the 50 liters harvested from the run, we evaluated depth filtration and compared the results to centrifugation. Multiple filter trains with different properties were tested and the results on recovery, turbidity and impurity reduction will be presented and discussed

Polymer microarrays rapidly identify competitive adsorbents of virus-like particles (VLPs)

The emergence of SARS-CoV-2 highlights the global need for platform technologies to enable rapid development of diagnostics, vaccines, treatments, and personal protective equipment (PPE). However, many current technologies require the detailed mechanistic knowledge of specific material-virion interactions before they can be employed, for example to aid in the purification of vaccine components, or in design of more effective PPE. Here we show that an adaption of polymer micro array method for screening bacterial-surface interactions allows for screening of polymers for desirable material-viron interactions. Non-pathogenic virus like particles including fluorophores are exposed to the arrays in aqueous buffer as a simple model of virons carried to the surface in saliva/sputum. Competitive binding of Lassa and Rubella particles is measured to probe the relative binding properties of a selection of copolymers. This provides the first step in the development of a method for discovery of novel materials with promise for viral binding, with the next being development of this method to assess absolute viral adsorption and assessment of the attenuation of the activity of live virus which we propose would be part of a material scale up step carried out in biological laboratory safety level 4 facilities and the use of more complex media to represent biological fluids

A Flow-Through Chromatographic Strategy for Hepatitis C Virus-Like Particles Purification

Biopharmaceuticals are currently becoming one of the fastest growing segments of the global pharmaceutical industry, being used in practically all branches of medicine from disease treatment to prevention. Virus-like particles (VLP) hold tremendous potential as a vaccine candidate due to their anticipated immunogenicity and safety profile when compared to inactivated or live attenuated viral vaccines. Nevertheless, there are several challenges yet to be solved in the development and manufacturing of these products, which ultimately can increase time to market. Suchlike virus-based products, the development of a platform approach is often hindered due to diversity and inherent variability of physicochemical properties of the product. In the present work, a flow-through chromatographic purification strategy for hepatitis C VLP expressed using the baculovirus-insect cell expression system was developed. The impact of operational parameters, such as residence time and ionic strength were studied using scaled-down models and their influence on the purification performance was described. The flow-through strategy herein reported made use of radial-flow chromatography columns packed with an anion exchanger and was compared with a bind and elute approach using the same chromatography media. Overall, by selecting the optimal operational setpoints, we were able to achieve higher VLP recoveries in the flow-through process (66% versus 37%) with higher removal of DNA, baculovirus and host-cell protein (92%, 99% and 50% respectively)