Multi-column chromatographic purification of influenza virus-like particles

Seasonal Influenza occurs all over the world and causes approximately 500 million cases of infection and up to 500 thousand deaths annually. The most effective way to prevent the disease is throughout vaccination. However, the constant antigenic drift on the influenza virus implies an annually vaccine update with high inherent costs. A new generation of virus-like particles (VLPs) vaccines, that have the ability to stimulate the production of broad antibody response to different Influenza strains, is a promising approach to solve this problem. VLPs have become a promising solution for influenza pandemics as well. The high attractiveness of VLPs has led to an increasing interest in the development of VLP purification processes as it often accounts for the major production costs. Therefore, it is mandatory to improve downstream processing (DSP) trains, not only to increase the efficiency of the existing processes but also to develop new unit operations capable of coping with the stringent regulatory requirements. One of the most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. The process herein described is based on the optimal scheduling of the operations steps characteristic of a single-column bind and elute operation such as equilibration, product application, production and regeneration applied to a train of two columns. In fact, a simple serial connection of two columns, during the product application step, directing the effluent of the first column in the train to an adjacent one, allows the capture of the mass transfer zone. This setup modification not only avoids product loss but also greatly increases the capacity utilization of the chromatographic media by achieving column saturation. We report on the development of a multi-column chromatographic process aimed at the purification of influenza VLPs, produced using insect cell-based expression with recombinant baculovirus. The inherent potential to improve process efficiency and economics, providing a powerful and flexible alternative to conventional batch chromatography will be demonstrated highlighting the impact of factors such as manufacturing scale, the complexity of the experimental setup and imposed product quality requirement

Advancing downstream purification of cell and gene therapy medicinal products

The advent of advanced therapies in the biopharmaceutical industry has moved the spotlight into complex products such as viral vectors or stem cells, holding great promise in a myriad of clinical targets. Currently, the challenge for a widespread application of these new biopharmaceuticals is the development of cost-effective bioprocesses while maintaining product\u27s bioactivity and quality attributes. This presentation will focus on the latest advancements on downstream purification of cell and gene therapy medicinal products, supported by process innovation and the flexibility of old, but robust technologies such as tangential flow filtration (TFF) and chromatography. Improvement of purification yields of virus based biopharmaceuticals can be achieved through the rational development of alternative strategies, combining different modes of operation, such as flow through purification or multi-column chromatography, together with the recent developments of chromatographic media and fundamental understanding of the adsorption phenomena as reported for the case of gene therapy medicinal products. Critical quality attributes of cell based medicinal products cannot be compromised by the processing route chosen. The use of the already established TFF technology has the potential to improve the purity of cell based products, with the evaluation of critical process parameters of cell concentration and washing being of paramount importance. The purity of such products can also be incremented with the use of negative mode expanded bed adsorption chromatography with a new multimodal prototype matrix based on core–shell bead technology as demonstrated for the case of human mesenchymal stem cells. In summary, the advancement of the purification of complex biopharmaceutical entities, such as the ones here reported, can be described as an incremental process, but still with space for inn

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

Universal and in-process analytical tool for Influenza quantification using a label-free technology

Virus-like particles (VLPs) have become a promising solution for influenza pandemics, leading to an increasing interest on the development of VLP purification processes. However, the analytical methods used to detect and quantify VLPs are not yet able to keep up with the downstream progress. Currently, quantification relies on traditional methods such as hemagglutination (HA) assay, Single Radial Immunodiffusion (SRID) assay or Neuraminidase (NA) enzymatic activity assays. However, these analytical technologies are time-consuming, cumbersome and are only reliable for final product quantification and characterization, posing challenges for efficient downstream process development and monitoring. [1] Here we report a label-free tool that uses Biolayer interferometry (BLI) technology applied on an octet platform to detect and quantify Influenza VLPs at all stages of downstream processing (DSP). Human (α2,6-linked sialic acid) and avian (α2,3-linked sialic acid) biotinylated receptors associated with streptavidin biosensors were used, in order to quantify HA content [2] in several mono- and multivalent Influenza VLP strains. The applied method was able to detect and quantify HA from crude sample up to final VLP product. The resulting concentration values are similar to HA quantification method. BLI technology showed promising results as a high throughput analytical method with high accuracy and improved detection limits, when compared to traditional approaches. Moreover, it eliminates the need of fresh erythrocytes and reduces user variations on the quantitation. This simple and fast tool allowed for robust real-time results, which is crucial for in-line monitoring of DSP. Since the main goal of the work performed is to improve process control as well as monitoring, it may be used as a PAT (process analytical technology) tool. [1] THOMPSON et al., Virology Journal, 10:141, 2013. [2] CRUSAT et al., Virology, 447, 326-337, 2013

Addressing the challenges of influenza virus-like particles purification

Virus-like particles (VLPs) have been widely used in vaccine development over the last decades [1]. In fact, there are already several approved human vaccines against viruses that use recombinant VLPs as antigen, e.g. for hepatitis B virus and human papillomavirus [2]. Vaccination remains the most effective way to prevent infection with influenza viruses. However, their constant antigenic drift requires an annual update of the seasonal vaccine to prevent influenza epidemics [3-4]. To use the full potential of VLPs as vaccines efficient upstream processing as well as downstream processing (DSP) trains need to be established. The latter is of particular importance as it often accounts for the major biomanufacturing costs. Here we describe the establishment of an improved DSP unit train platform, adapted from virus particles to influenza VLPs, using pseudo-affinity sulfated cellulose membrane adsorbers (SCMA) [5]. An initial clarification step prepares the bulk for the subsequent purification steps. SCMA performance was optimized using a design of experiments (DoE) approach. More than 80% of the product was recovered with removal of host cell protein and DNA above 89% and 80%, respectively. This represents a significant improvement in performance compared to the traditional use of ion exchangers commercially available. Using this SCMA platform for influenza virus particles purification we were able to speed up the process by decreasing the number of DSP steps, to improve the scale-up and to reduce costs due to the removal of other chromatographic steps. References [1] L. Lua, et al., Biotechnology and Bioengineering, 111(3): p. 425-440 (2014). [2] Q. Zhao, et al., Trends in Biotechnology, 31(11): p. 654-663 (2013). [3] D. Smith, et al., Science, 305(5682): p. 371-376 (2004). [4] C. Thompson, et al., Virology Journal, 10 (2013). [5]M. Wolff, and U. Reichl, Expert Review of Vaccines, 10(10): p. 1451-1475 (2011)

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

Conventional and alternative antifungal therapies to oral candidiasis

Candida-associated denture stomatitis is the most common form of oral candidal infection, with Candida albicans being the principal etiological agent. Candida adheres directly or via an intermediary layer of plaque-forming bacteria to denture acrylic. Despite antifungal therapy to treat denture stomatitis, infection is reestablished soon after the treatment ceases. In addition, many predisposing factors have been identified as important in the development of oral candidiasis, including malnourishment, common endocrine disorders, such as diabetis mellitus, antibacterial drug therapy, corticosteroids, radiotherapy and other immunocompromised conditions, such as acquired immunodeficiency syndrome (AIDS). These often results in increased tolerance to the most commonly used antifungals. So this review suggests new therapies to oral candidiasis.82483