Wang Chunlei, Mulagapati Sri Hari Raju, Chen Zhongying, Du Jing, Zhao Xiaohui, Xi Guoling, Chen Liyan, Linke Thomas, Gao Cuihua, Schmelzer Albert, Liu Dengfeng
Molecular Therapy Methods & Clinical Development, Volume 15, September 26 2019, Pages 257-263
Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 1011 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.
Keywords: AAV, AAV6.2, Chromatography, Anion exchange chromatography (AEC), Empty capsids, AUC, High-throughput
Sofiya Fedosyuk, Thomas Merritt, Marco Polo Peralta-Alvarez, Susan J. Morris, Ada Lam, Nicolas Laroudie, Anilkumar Kangokar, Daniel Wright, George M. Warimwe, Phillip Angell-Manning, Adam J. Ritchie, Sarah C. Gilbert, Alex Xenopoulos, Anissa Boumlic, Alexander D. Douglas
Published online 30 April 2019.
A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.
Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell – promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype.
Keywords: Simian adenovirus, GMP, Clinical trials, Single-use, Biomanufacturing, Bioreactor, Purification
Dr. Xiaotong Fu, Dr. Wei-Chiang Chen, C. Argento, R. Dickerson, P. Clarner, V. Bhatt, G. Bou-Assaf, Dr. M. Bakhshayeshi, Dr. Xiaohui Lu, Dr. S. Bergelson, Dr. J. Pieracci
Human Gene Therapy (2019)
Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids—viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography (AEX-HPLC) assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability to both the transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC) methods used in empty-to-full capsid ratio quantification, yet providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes (vg)/ml.
K. Trabelsi, M. Ben Zakour, H. Kallel
Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease.
Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield.
Laura M. Fischer, Michael W. Wolff, Udo Reichl, Vaccine 2017 July 17
The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA. The continuous chromatography was implemented as simulated moving bed chromatography (SMB) in a three zone open loop configuration with a detached high salt zone for regeneration.
SMBs exploiting 10% and 50% of the monoliths’ dynamic binding capacity, respectively, allowed the depletion of >98% of the DNA and >52% of the total protein. Based on the hemagglutination assay (HA assay), the virus yield was higher at 10% capacity use (89% vs. 45%). Both SMB separations resulted in a ratio of total protein to hemagglutinin antigen (based on single radial diffusion assay, SRID assay) below the required levels for manufacturing of human vaccines (less than 100 mg of protein per virus strain per dose). The level of contaminating DNA was five-times lower for the 10% loading, but still exceeded the required limit for human vaccines. A subsequent Benzonase treatment step, however, reduced the DNA contamination below 10 ng per dose. Coupled to continuous cultivations for virus propagation, the establishment of integrated processes for fully continuous production of vaccines seems to be feasible.
Tsutomu Arakawa, Pete Gagnon
Journal of Pharmaceutical Sciences 107 (2018) 2297-2305
The concept of cosolvent exclusion was developed by a group of Timasheff's laboratory in 1970-1990 and is currently used widely to explain the effects of a variety of cosolvents on the stability and solubility of macromolecules. Not surprisingly, these concepts have had substantial influence in the fields of formulation, protein folding and unfolding, but they have perhaps more surprisingly found their way into the field of chromatography. A variety of excluded cosolvents have been used to enhance binding and resolution of proteins and other macromolecules in ion exchange, hydroxyapatite, affinity, and hydrophobic interaction chromatography. These cosolvents include salting-out salts, amino acids and polymers, and frequently polyethylene glycol (PEG). A new mode of chromatography, termed “steric exclusion chromatography,” was recently introduced. It employs hydroxylated solid phase surfaces. Steric exclusion of the PEG stabilizes the association of macromolecules with the solid phase. Elution is achieved by reducing the PEG concentration. Magnetic particles are also used in this chromatography. This review summarizes the concepts of preferential cosolvent exclusion and its applications in column chromatography.
V.Rajamanickam, D.Wurm, C.Slouka, C.Herwig, O.Spadiut
Anal Bioanal Chem (2016)
The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future
Alicia T Lucero, Sergio A Mercado, Anamaría C Sánchez,Carolina A Contador, Barbara A Andrews and Juan A Asenjo, Journal of chemical technology and biotechnology, (2017)
BACKGROUND: Gene therapy is a potent alternative for long-lasting inhibition of alcohol consumption. This study compares the purification of a recombinant adenoviral vector serotype 5 (rAdV5) for use in gene therapy against alcoholism using two anion-exchange methods.
RESULTS: Two anion-exchange chromatography methods using fast protein liquid chromatography were compared using a packed-bed column (Q-Sepharose™ XL) and two monolithic columns (CIM™ QA-1 and CIM™ DEAE-1). An improved and reproducible separation of recombinant adenovirus type 5 from cell lysate contaminants was achieved using the two strong anion-exchange columns in a two-step gradient chromatography. Higher adenovirus yields were achieved using the CIM QA-1 tube monolithic column at sample volumes of both 1 and 10 mL compared with the Q-Sepharose XL column. At higher flow rates, the CIM QA-1 tube monolithic column achieved better separation of the target fraction. Process recovery was improved from 28% using the Q-Sepharose XL column to 34% with the CIM QA-1 tube monolithic column quantified as vector genome. Analysis by SDS-PAGE demonstrated a purity of 70% for purified adenovirus using the CIM QA-1 tube monolithic column.
CONCLUSION: This study indicated that the use of a CIM QA-1 tube monolithic column is a better alternative than Q-Sepharose XL, and CIM DEAE-1 tube monolithic columns for the primary purification process of rAdV5 carrying the human aldehyde dehydrogenase-2 antisense gene. This purification strategy has been used as a basis to scale-up a GLP process for the production of material at the National Research Council of Canada to be used in preclinical trials of this gene therapy against alcoholism
Sebastijan Peljhan, Tina Jakop, Dunja Šček, Vid Skvarča, Blaž Goričar, Romina Žabar, Nina Mencin. Electrophoresis 2017 July 20
The plasma-derived IgG used either for diagnostic purpose or intravenous application (in form of IVIG) in various medical therapies is certainly gaining more and more attention on annual basis. Different manufacturing processes are used to isolate immunoglobulins from human plasma. However, a quest for alternative paths in IgG isolation not only requires development of the most efficient isolation process, but also a rapid and reliable analytics to track the purification. Fast and reliable fingerprint based method for characterization of IgG prepared from Cohn I+II+III paste is presented in this paper. The fingerprint method bases on partial separation of proteins in linear gradient on CIMacTM quaternary amine, strong anion exchange group (QA) 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples during the IgG isolation from Cohn I+II+III fraction paste, but very accurate qualitative information about the composition of the sample can be obtained in less than 5 min. From the differences in the chromatograms of various samples, the ratio between IgG and impurities in each sample can be easily assessed. The method is suitable for input material control, in-line monitoring of the downstream processing, final control of the products, as well as in stability studies and enables taking fast and accurate decisions during fractionation process.
David Vincent, Petra Kramberger, Rosana Hudej, Aleš Štrancar, Yaohe Wang,Yuhong Zhou, Ajoy Velayudhan
The purification of large viruses remains an important field of research and development. The development of efficient purification trains is limited by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8μm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths. Without the need for nuclease treatment, up to 99% of the total DNA loaded can be removed from the vaccinia feed stream by the CIM OH monolith, which also reduces the total protein concentration in the product pool to LLOQ levels, and achieves infectious virus recoveries of 90%. Binding capacities of greater than 1x109 pfu of vaccinia per mL of matrix were obtained on both CIM SO3 and CIM OH monoliths. Multiple orthogonal analytical methods have been used to develop process knowledge and understanding.
Antonio M. Munoz, Paul Yourik, Vaishnavi Rajagopal, Jagpreet S. Nanda, Jon R. Lorsch, Sarah E. Walker
RNA Biology, 2017, VOL. 14, NO. 2, 188–196
In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.
P. Stepperta, D. Burgstallera, M. Klausbergera, E. Bergerb, P.P. Aguilara, T.A. Schneiderb, P. Krambergerc, A. Toverd, K. Nöbauere, E. Razzazi-Fazelie, A. Jungbauer, Journal of Chromatography A, 1455 (2016)
Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification ofVLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scaleable purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHOcells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed throughthe column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA waseluted prior to VLPs and particles in the range of 100–200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysisin this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×109 particles, could be processed witha 1mL monolith within 47 min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.
J. Transfiguracion, A. P. Manceur, E. Petiot, C. M. Thompson, A. A. Kamen
The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris–HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 109 and 1 × 1011 virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 108 and 4.35 × 109 whereas the limit of quantification was between 6.90 × 108 and 1.45 × 1010 VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.
D. Buzzi, A. Štrancar
Chimica Oggi-Chemistry Today; Vol 33(1) January/February 2015
The importance of the monitoring of a process all along its steps by means of PAT has been defined by FDA in 2002. How can be defined the product quality and what are the parameters that should be checked by means of different analysis techniques, being focused in particular on the application of high pressure liquid chromatography techniques (HPLC) as high value tool for the process monitoring. From the first introduction of Process Analytical Technology to the "state of the art": how can be PAT implemented in order to ensure the final product quality.
J. Ruscic, I. Gutiérrez-Aguirre, M. Tusek Znidaric, S. Kolundzija, A. Slana, M. Barut, M. Ravnikar, M. Krajacic
Journal of Chromatography A, 1388 (2015) 69–78
The emergence of next-generation "deep" sequencing has enabled the study of virus populations with much higher resolutions. This new tool increases the possibility of observing mixed infections caused by combinations of plant viruses, which are likely to occur more frequently than previously thought. The bio-logical impact of co-infecting viruses on their host has yet to be determined and fully understood, and the first step towards reaching this goal is the separation and purification of individual species. Ion-exchange monolith chromatography has been used successfully for the purification and concentration of different viruses, and number of them have been separated from plant homogenate or bacterial and eukaryoticlysate. Thus, the question remained as to whether different virus species present in a single sample could be separated. In this study, anion-exchange chromatography using monolithic supports was optimized for fast and efficient partial purification of three model plant viruses: Turnip yellow mosaic virus, Tomato bushy stunt virus, and Tobacco mosaic virus. The virus species, as well as two virus strains, were separated from each other in a single chromatographic experiment from an artificially mixed sample. Based on A260/280 ratios, we were able to attribute specific peaks to a certain viral morphology/structure (icosa-hedral or rod-shaped). This first separation of individual viruses from an artificially prepared laboratory mixture should encourage new applications of monolithic chromatographic supports in the separation of plant, bacterial, or animal viruses from all kinds of mixed samples.
P. Kramberger, U. Lidija, A. Štrancar
Human Vaccines & Immunotherapeutics, 11:4 (2015) 1010-1021
Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production.
M. Zaveckas, S. Snipaitis, H. Pesliakas, J. Nainys, A. Gedvilaite
Journal of Chromatography B, 991 (2015) 21–28
Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impacton swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccinecandidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizeswere examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. QSepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some hostcell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6Band CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPsafter chromatography on Heparin Sepharose CL-6B was only 4–7% and the recovery of VLPs was 5–7%.Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purityof about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 mono-lith was 15–18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electronmicroscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate wasdeveloped using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith.
A. G. Lopes
FBP-461, Food and Bioproducts Processing (2014)
As the biopharmaceutical industry matures, the trend towards increased flexibility and productivity, faster time tomarket and greater profitability are driving the replacement of traditional stainless steel equipment by single-use technology (SUT). The use of SUT in the biopharmaceutical industry can significantly impact the manufacturing process efficiency by reducing capital costs, improving plant flexibility, reducing start-up times and costs, and elim-inating both non-value added process steps and the risk of cross-contamination. In addition it significantly reduces process liquid waste, labour costs and on-site quality and validation requirements. This paper reviews the current status of the technology and the impact of SUT in the biopharmaceutical industry, with the aim of identifying the challenges and limitations that still need to be addressed for further adoption of these technologies. Even tough SUT has a multitude of systems available, its components and assemblies have little standardisation as well as alack of harmonised tests and procedures among suppliers, with an array of guidelines from a variety of sourcesand no critical limits have been established. In addition, the use of SUT has new validation requirements such as leachables and extractables, suppliers’ qualification and SUT lot-to-lot variability. The lack of expertise in these areas and the new training requirements when using SUT also need to be addressed. To date the majority of the avail-able literature regarding SUT is found in trade journals where typically suppliers are the main contributors. There is still a lack of engagement of the academic community, which contributes to very limited scientific proof from independent peer-reviewed research to support performance of SUT. This is particularly the case during operation and integrity testing of SUT, during for example on-site testing, transport and disposal. Another area where no work has been undertaken concerns conceptual approaches for facility clean-room requirement and appropriate layout design using SUT. Investment in novel technologies, research, standardisation and training is paramount for further development and implementation of SUTs across all sectors of the biopharmaceutical industry.
M. M. Segura, M. Puig, J. Piedra, S. Miravet
Adenovirus: Methods and protocols, Methods in Molecular Biology, vol. 1089
Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration–diafiltration. A Benzonase® digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38–45 %.
M.-C. Claudepierrea, et al.
Journal of Virology, February 2014
To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293-cells lines, double-positive for human Toll-like receptors (TLR) and a NF-κB-inducible reporter gene. Screening a large variety of compounds and cellular extracts detected a TLR3 activating compound in a microsomal yeast extract. Fractionation of this extract identified a RNA molecule of 4.6 kb, named Nucleic Acid Band 2 (NAB2) that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to the genome of the dsRNA L-BC virus of Saccharomyces cerevisiae. A large scale production and purification process of this RNA was established based on chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin, but neither NAB2 nor Lipofectin alone, induced the secretion of IL-12(p70), IFNα, IP-10, Mip-1β and IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 signaled also via the cytoplasmic sensor for RNA recognition MDA-5. Significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding MVA and NAB2+Lipofectin. This combination of immunotherapeutics strongly increased the percentage of infiltrating Natural Killer (NK) cells and plasmacytoid dendritic cells (pDCs) at the injection sites, cell types which can modulate innate and adaptive immune responses.