Publications
2015
J. Transfiguracion, A. P. Manceur, E. Petiot, C. M. Thompson, A. A. Kamen
Vaccine (2014)
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.
2014
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
Vaccine 2014
We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruses on 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 four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the 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-H1N1 virus 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 virus on CIM QA were between 70.8±32.3% and 87±30.8%. Total protein removal varied from 93.3±0.4% to 98.6±0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease.
2013
A. A. Shukla, U. Gottschalk
Trends in Biotechnology (2012) 1-8
The manufacture of protein biopharmaceuticals is conducted under current good manufacturing practice (cGMP) and involves multiple unit operations for upstream production and downstream purification. Until recently, production facilities relied on the use of relatively inflexible, hard-piped equipment including large stainless steel bioreactors and tanks to hold product intermediates and buffers. However, there is an increasing trend towards the adoption of single-use technologies across the manufacturing process. Technical advances have now made an end-to-end single-use manufacturing facility possible, but several aspects of single-use technology require further improvement and are continually evolving. This article provides a perspective on the current state-of-the-art in single-use technologies and highlights trends that will improve performance and increase the market penetration of disposable manufacturing in the future.
M. Li, Y. X. Qiu
Vaccine 31 (2013) 1264-1267
An effective downstream bio-processing of vaccine products requires complete chemical knowledge of the contaminants that may arise from a given vector expression system. Whether the vaccine is made from the traditional egg-based or the new cell-cultured process, it is the expression system that determines the types of impurities that need to be identified and removed from the vaccine product.
There are mechanical and chemical factors that can either reduce the yield or render a vaccine product to be irreversibly inactive. The choice of equipment and solvents is therefore important in minimizing product loss, and for maintaining an efficient and optimized manufacturing process.
The frequent out-of-specification, irreproducible data and inefficiency in the manufacturing of biologics were the basis for FDA to propose the “cGMP for the 21st Century” initiative in the year of 2000. Effective 2004, the concept of quality by design (QbD) has been imposed in the manufacturing of biologics. To facilitate the implementation of QbD FDA has encouraged the use of process analytical technology (PAT). Further, FDA believes that an optimized manufacturing scheme requires one to identify and to control the variables that can negatively affect the yield and quality of the desired product, and PAT can reveal wrongful data and alert the operator for immediate correction during processing.
P. Fernandes, C. Peixoto, VM Santiago, EJ Kremer, AS Coroadinha and PM Alves
Gene Therapy (2012), 1–8
Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors producedin MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of ~109 infectious particles (IP) ml-1 and 2x103 IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.
Attachments
P. Gerster, E.-M. Kopecky, N. Hammerschmidt, M. Klausberger, F. Krammer, R. Grabherr, C. Mersich, L. Urbas, P. Kramberger, T. Paril, M. Schreiner, K. Nöbauer, E. Razzazi-Fazeli, A. Jungbauer
Journal of Chromatography A, 1290 (2013) 36-45(2013) 36-45
A chromatographic process based on monoliths for purification of infective baculovirus without prior concentration step has been established. Baculovirus produced in Spodoptera frugiperda cells (Sf-9) were harvested by centrifugation, filtered through 0.8 μm filters and directly loaded onto radial 1 mL anion exchange monoliths with a channel size of 1.5–2.0 μm operated at a volumetric flow rate of one bed volume per minute. Optional an epoxy monolith was used as pre-column to reduce interfering compounds and substances influencing the capacity of anion exchange monoliths for baculovirus infectious virus could be eluted with a step gradient at salt concentrations of 440 mM NaCl. Recovery of infectious virus was highly influenced by composition and age of supernatant and ranged from 20 to >99% active baculovirus. Total protein content could be reduced to 1–8% and DNA content to 38–48% in main virus fraction. Infective virus could be 52-fold concentrated within 20.5 h and simultaneously an 82-fold volume reduction was possible when loading 1150 mL (2.1 × 108 pfu/mL) onto 1 mL scale support.
A. Steyer, I. Gutierrez-Aguire, M. Kolenc, S. Koren, D. Kutnjak, M. Pokorn, M. Poljšak-Prijatelj, N. Rački, M. Ravnikar, M. Sagadin, A. Fratnik Steyer, N. Toplak
Journal of Clinical Microbiology, November 2013
Mammalian orthoreoviruses (MRV) are known to cause mild enteric and respiratory infections in humans. They are widespread and infect a broad spectrum of mammals. We report here the first case of MRV detected in a child with acute gastroenteritis, which showed the highest similarity to MRV reported recently in European bats. Stool sample examination of the child was negative for most common viral and bacterial pathogens. Reovirus particles were identified by electron microscopic examination of both stool suspension and cell culture supernatant. The whole genome sequence was obtained with the Ion Torrent next generation sequencing platform. Prior to sequencing, stool sample suspension and cell culture supernatant were pre-treated with nucleases and/or the convective interaction media (CIM) monolithic chromatographic method to purify and concentrate the target viral nucleic acid. Whole genome sequence analysis revealed that the Slovenian SI-MRV01 isolate was most similar to MRV found in bat in Germany. High similarity was shared in all genome segments, with nucleotide and amino acid identities between 93.8-99.0% and 98.4-99.7%, respectively. It was shown that CIM monolithic chromatography alone is an efficient method for enriching the sample in viral particles before nucleic acid isolation and next generation sequencing application.
E. Maksimova, E. Vlakh, E. Sinitsyna, T. Tennikova
J. Sep. Sci. 2013, 36, 3741–3749
Ultrashort monolithic columns (disks) were thoroughly studied as efficient stationary phases for precipitation–dissolution chromatography of synthetic polymers. Gradient elution mode was applied in all chromatographic runs. The mixtures of different flexible chain homopolymers, such as polystyrenes, poly(methyl methacrylates), and poly(tert-butylmethacrylates) were separated according to their molecular weights on both commercial poly(styrene-co divinylbenzene).
disks (12 id × 3 mm and 5 × 5 mm) and lab-made monolithic columns (4.6 id × 50 mm) filled with supports of different hydrophobicity. The experimental conditions were optimized to reach fast and highly efficient separation. It was observed that, similar to the separation of monoliths of other classes of (macro)molecules (proteins, DNA, oligonucleotides), the length of column did not affect the peak resolution.
A comparison of the retention properties of the poly(styrene-co-divinylbenzene) diskshaped monoliths with those based on poly(lauryl methacrylate-co-ethylene dimethacrylate), poly(butyl methacrylate-co-ethylene dimethacrylate), and poly(glycidyl methacrylate-co-ethylene dimethacrylate) supports demonstrated the obvious effect of surface chemistry on the resolution factor. Additionally, the results of the discussed chromatographic mode on the fast determination of the molecular weights of homopolymers used in this study were compared to those established by SEC on columns packed with sorbent beads of a similar nature to the monoliths.
Roy N D‘Souza, Ana M Azevedo, M Raquel Aires-Barros, Nika Lendero Krajnc, Petra Kramberger, Maria Laura Carbajal, Mariano Grasselli, Roland Meyer & Marcelo Fernández-Lahore
Vol. 1, No. 5, Pharmaceutical Bioprocessing (2013)
Downstream processing is currently the major bottleneck for bioproduct generation. In contrast to the advances in fermentation processes, the tools used for downstream processes have struggled to keep pace in the last 20 years. Purification bottlenecks are quite serious, as these processes can account for up to 80% of the total production cost. Coupled with the emergence of new classes of bioproducts, for example, virus-like particles or plasmidic DNA, this has created a great need for superior alternatives. In this review, improved downstream technologies, including aqueous two-phase systems, expanded bed adsorption chromatography, convective flow systems, and fibre-based adsorbent systems, have been discussed. These adaptive methods are more suited to the burgeoning downstream processing needs of the future, enabling the cost-efficient production of new classes biomaterials with a high degree of purity, and thereby hold the promise to become indispensable tools in the pharmaceutical and food industries.
2012
J. Lee, H. T. Gan, S. M. Abdul Latiff , C. Chuah, W. Y. Lee, Y.-S. Yang, B. Loo, S. K. Ng, P. Gagnon
Journal of Chromatography A, 1270 (2012) 162-170
We introduce a chromatography method for purification of large proteins and viruses that works by capturing them at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG). No direct chemical interaction between the surface and the target species is required. We refer to the technique as steric exclusion chromatography. Hydroxyl-substituted polymethacrylate monoliths provide a hydrophilic surface and support convective mass transport that is unaffected by the viscosity of the PEG. Elution is achieved by reducing PEG concentration. Selectivity correlates with molecular size, with larger species retained more strongly than smaller species. Retention increases with PEG size and concentration. Salts weaken retention in proportion to their concentration and Hofmeister ranking. Retention is enhanced near the isoelectric point of the target species. Virus binding capacity was measured at 9.9 × 1012 plaque forming units per mL of monolith. 99.8% of host cell proteins and 93% of DNA were eliminated. Mass recovery exceeded 90%. IgM capacity was greater than 60 mg/mL. 95% of host cell proteins were eliminated from IgM produced in protein-free media, and mass recovery was up to 90%. Bioactivity was fully conserved by both viruses and antibodies. Process time ranged from less than 30 min to 2 h depending on the product concentration in the feed stream.
H. M. Oksanen, A. Domanska, D. H. Bamford
Virology Volume 434, Issue 2, 20 December 2012
We report anion exchange chromatographic purification method powerful for preparation of virus particles with ultra pure quality. The technology is based on large pore size monolithic anion exchangers, quaternary amine (QA) and diethylaminoethyl (DEAE). These were applied to membrane-containing icosahedral bacteriophage PRD1, which bound specifically to both matrices. Virus particles eluted from the columns retained the ir infectivity, and were homogenous with high specific infectivity. The yields of infectious particles were up to 80%. Purified particles were recovered at high concentrations, approximately 5mg/ml, sufficient for virological, biochemical and structural analyses. We also tested the applicability of the monolithic anion exchange purification on a filamentous bacteriophage phi 05_2302. Monolithic ion exchange chromatography is easily scalable and can be combined with other preparative virus purification methods.
P. Gagnon
Journal of Chromatography A, 1221 (2012) 57-70(2012) 57-70
This article reviews technology trends in antibody purification. Section 1 discusses non-chromatography methods, including precipitation, liquid–liquid extraction, and high performance tangential flow filtration. The second addresses chromatography methods. It begins with discussion of fluidized and fixed bed formats. It continues with stationary phase architecture: diffusive particles, perfusive particles, membranes and monoliths. The remainder of the section reviews recent innovations in size exclusion, anion exchange, cation exchange, hydrophobic interaction, immobilized metal affinity, mixed-mode, and bioaffinity chromatography. Section 3 addresses an emerging trend of formulating process buffers to prevent or correct anomalies in the antibodies being purified. Methods are discussed for preventing aggregate formation, dissociating antibody-contaminant complexes, restoring native antibody from aggregates, and conserving or restoring native disulfide pairing.
M. Žorž
ChemieXtra 3/2012 pp 30-33
Sartorius BIA Separations produziert und vertreibt kurze monolithischen Chromatografiesäulen, die auf der CIM-Convective Interaction Media-Technologie basieren. CIM-Säulen eignen sich vor allem für die Reinigung von grossen Biomolekülen wie etwa Viren (virale Vektoren und Impfstoffe), DNA (Plasmid-DNA) und grössere Proteine (Immunglobuline G und M, pegylierte Proteine). Sie weisen einzigartige Eigenschaften in Bezug auf operative Flussraten, Adsorptionsfähigkeit und Trennung grosser Biomoleküle auf. Die Säulen werden in Forschung, Labor, Pilot- und industriellen Produktionsstufen eingesetzt und sind extrem einfach zu handhaben.