Pete Gagnon, Maja Leskovec, Sara Drmota Prebil, Rok Žigon, Maja Štokelj, Andrej Raspor, Sebastijan Peljhan, Ales Štrancar
Journal of Chromatography A, 2021
Separation of empty and full adeno-associated virus capsids by multimodal metal affinity chromatography was investigated using a positively charged metal affinity ligand. A subpopulation of empty capsids eluted first, followed by full capsids, and later by more empty capsids and debris. Empty and full capsid composition of chromatography fractions was evaluated by cesium chloride density gradient centrifugation followed by stratigraphic flow analysis of the centrifuge tube contents, monitored by intrinsic fluorescence. Columns charged with barium, calcium, magnesium, zinc, manganese, and ferric ions gave similar results with respect to capsid separation. Charging with cupric ions maintained resolution between early-eluting empty capsids and full capsids but caused them to elute at lower conductivity. Empty and full capsids were fractionated with Tris-borate gradients, sodium chloride gradients, and magnesium chloride gradients. Recovery of full serotype 9 capsids was 100% with complete elimination of empty capsids. All metal ions bound contaminant subsets that required sodium hydroxide for removal. Columns charged with ferric iron and manganese bound more contaminants than all other metals. Columns charged with calcium, magnesium, barium, and copper bound the least. Contaminant binding on zinc-charged columns was intermediate between the two groups.
Michael Winkler, Mikhail Goldfarb, Shaojie Weng, Jeff Smith, Susan Wexelblat, John Li, Alejandro Becerra, Sandra Bezemer, Kevin Sleijpen, Aleš Štrancar, Sara Primec, Romina Zabar, April Schubert, Akunna Iheanacho, and David Cetlin
BioProcess International, April 2021
Over the past decade, adenoassociated virus (AAV) vectors have become established as leading gene-delivery vehicles. In 2017, the pipeline for gene therapies included 351 drugs in clinical trials and 316 in preclinical development. As those candidates advance, significant efforts are being made in process development and manufacturing for viral vectors, with the overall goal of reducing process impurities while maintaining the highest possible process yield.
BIA Separations (now part of Sartorius AG) has developed and commercialized CIMmultus QA monoliths, which have been cited in several AAV downstream processes for their ability to separate empty and full virus particles effectively. Monolithic supports represent a unique type of stationary phase for liquid chromatography, bioconversion, and solid-phase synthesis. Aside from increased processing speed, monolithic flow-through pores (channels) also provide easy access for large molecules, which supports both purification and depletion of nanoparticles such as plasmid DNA (pDNA) molecules and AAV particles.
Sebastijan Peljhan, Maja Štokelj, Sara Drmota Prebil, Pete Gagnon and Aleš Štrancar
Cell & Gene Therapy Insights, March 2021
Ultracentrifugation (UC) is a well-known technique for fractionating adeno-associated virus (AAV) capsids according to their density, which is mainly a function of their encapsidated DNA mass. Empty capsids represent the lowest density subpopulation. Full capsids represent the highest density subpopulation, sometimes accompanied by partially full capsids of intermediate density. Fractions can be collected after sedimentation for analysis but the practice is laborious and discourages application of multiple monitoring techniques that might provide deeper insights into sample composition. Anion exchange chromatography (AEC) also achieves fractionation of empty and full capsids for many AAV serotypes. The degree of separation varies among serotypes and does not correlate strictly with UC. This is not surprising since separation by AEC is highly influenced by capsid surface charge, which is independent of the amount of DNA packaged within the capsids. Chromatography methods however present a significant analytical advantage in the ease of monitoring the column effluent, including with multiple detectors. UV absorbance at 260 nm and 280 nm permits estimation of empty and full capsid proportions in any given peak. Intrinsic fluorescence enables estimation of relative areas of empty capsid peaks and full capsid peaks. Light scattering does the same and permits the further determination of capsid size and mass. In this report, we merge UC with an HPLC monitoring array to simultaneously analyze dual wavelength UV, intrinsic fluorescence, and light scattering through cesium chloride density gradient strata. Limitations of each monitoring method are discussed. UC results are compared with chromatography profiles to highlight distinction between separation methods. Practical application of results for final product characterization is considered, along with potential to support development of better purification processes.
Pete Gagnon, Maja Leskovec, Blaz Goricar, and Aleš Štrancar
BPI, December 17, 2020
With its first licensed therapeutic now marketed worldwide, adeno-associated virus (AAV) has become a preferred vector for gene therapy. However, unlocking its full potential still poses challenges, many of which are associated with purification. The first involves the transition from upstream to downstream processes. AAV-bearing lysates are laden with debris that foul filtration media and limit or prevent concentration. Another challenge involves reduction of soluble host-cell DNA, which is complicated by its strong association with nucleoproteins. A third involves elimination of empty capsids. Currently, ultracentrifugation meets that need, but scale-up issues make chromatographic alternatives attractive. A fourth challenge involves the need for rapid, accurate, and revealing analytical results to guide process development, support validation, document control, and enable reproducibility of manufacturing processes. The following article shares experimental data showing how those challenges can be addressed to advance the evolution of gene therapy with AAV.
Pete Gagnon, Blaz Goricar, Nina Mencin, Timotej Zvanut, Sebastijan Peljhan, Maja Lescovec and Ales Strancar
Pharmaceutics. 2021 Jan 17;13(1):113
HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.
by Maribel Rios, Aleš Štrancar, J. Michael Hatfield and Pete Gagnon
BioProcess International, 2020
Adenoassociated viral (AAV) vectors have become synonymous with gene therapy delivery. However, because they are produced in such small quantities and because their upstream processes carry comparatively large amounts of host-cell DNA and other impurities, AAV purification can be challenging. Several researchers have applied different chromatographic strategies, but no universal method has been adopted in the biopharmaceutical industry.
This eBook features a discussion among several industry experts that explores challenges specific to AAV purification, shedding light on whether current strategies and separation technologies are up to the task. The conversation traverses issues relating to material handling at the upstream–downstream interface, removal of host-cell DNA, chromatographic separation of empty and full capsids, and a lack of fast and robust in-process analytics for downstream processes. Participants also explore whether the rise of AAV-based treatments will require downstream scientists to shift away from the antibody-centered conceptions of chromatography that have grown alongside the biotherapeutics industry.
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.
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
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.
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.
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.
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.
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.
M. Banjac, E. Roethl, F. Gelhart, P. Kramberger, B. Lah Jarc, M. Jarc, A. Štrancar, T. Muster, M. Peterka
We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruseson ion exchange methacrylate monoliths. Influenza A ΔNS1-H1N1, ΔNS1-H3N2, ΔNS1-H5N1 and ΔNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All fourvirus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higherrecoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger atthe same pH, while ΔNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ΔNS1-H1N1virus were 1.9E + 10 TCID50/ml, 1.0E + 10 TCID50/ml and 8.9E + 08 TCID50/ml, respectively. Purification of ΔNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious viruson CIM QA were between 70.8 ± 32.3% and 87 ± 30.8%. Total protein removal varied from 93.3 ± 0.4% to98.6 ± 0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly dependedon pretreatment with deoxyribonuclease.