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

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

A click chemistry strategy to specifically monitor and improve purification of Influenza virus-like particles

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug-delivery. However, most applications use simple, non-enveloped VLPs that present less technical challenges, not only to produce and purify, but also in terms of characterization, compared to enveloped VLPs. Recent advances in upstream processing, new product quality requirements and other regulatory issues, as well as the search for more cost-effective processes, led to the need to develop more efficient downstream processes for biopharmaceuticals.[1] In that sense, new monitoring and product characterization methods, which can be applied at all stages of downstream processing, are needed. Here is reported a valuable platform for the downstream processing and monitoring of the in vivo production of site-specifically functionalized enveloped Influenza VLPs. This strategy involves a two-step procedure that consists of residue-specific replacement of methionine by an analog (azidohomolanine) that enabled for post-expression functionalization with a fluorophore.[2] Importantly, this platform does not impact VLP production or purification processes, and allows functionalization without deleterious effect on hemagglutinin biological function. As a proof of concept a complete downstream processing was performed, including clarification, capture and polishing steps. A flow cytometry analysis (FACS) step was added to achieve a refined discrimination and separation between VLPs and baculovirus - the major impurity of the process.[3] This was further confirmed using atomic force microscopy (AFM). This tool allowed to accurately monitor our product, achieve higher product recovery yields and higher impurity removal levels. The versatile system presented here is broadly applicable to the production of functionalized enveloped VLPs, for vaccine design, targeted drug delivery and molecular imaging. [1] CRAMER et al., Curr Opin Chem Eng, 1, 27-37, 2011. [2] BANERJEE et al., J. Virol., 85, 7546-7554, 2011. [3] VAN DER VLIST et al., Nature Protoc, 7.7, 1311-1326, 2012

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

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)

Clinical-grade oncolytic adenovirus purification using polysorbate 20 as an alternative for cell lysis

PD/BD/114034/2015Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives. Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorbate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impurities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell proteins and ds-DNA was 99.9% and 97.1%, respectively. Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the developed process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.publishersversionpublishe