Publications
2014
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
E. Mota, A. Sousa, U. Černigoj, J. A. Queiroz, C. T. Tomaz, F. Sousa
Journal of Chromatography A (2013)
The demand for high-purity supercoiled plasmid DNA to be applied as a vector for new therapeutic strategies, such as gene therapy or DNA vaccination has increased in the last years. Thus, it is necessary to implement an analytical technique suitable to control the quality of the supercoiled plasmid as a pharmaceutical product during the manufacturing process. The present study describes a new methodology to quantify and monitor the purity of supercoiled plasmid DNA by using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows the separation of the plasmid isoforms by combining a NaCl stepwise gradient. The specificity, linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a supercoiled plasmid concentration up to 200 μg/mL. The main advantage achieved by using this monolithic column is the possibility to quantify the supercoiled plasmid in a sample containing other plasmid topologies, in a 4 min experiment. This column also permits the assessment of the supercoiled plasmid DNA present in more complex samples, allowing to control its quality throughout the bioprocess. Therefore, these findings strengthen the possibility of using this monolithic column associated with a powerful analytical method to control the process development of supercoiled plasmid DNA production and purification for therapeutic applications.
S. Haberl, M. Jarc, A. Štrancar, M. Peterka, D. Hodžić, D. Miklavčič
J Membrane Biol, DOI 10.1007/s00232-013-9580-5
The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.
B. Gabor, U. Černigoj, M. Barut, A. Štrancar
Journal of Chromatography A, 1311 (2013) 106-114
HPLC based analytical assay is a powerful technique that can be used to efficiently monitor plasmid DNA (pDNA) purity and quantity throughout the entire purification process. Anion exchange monolithic and non-porous particle based stationary phases were used to study the recovery of the different pDNA isoforms from the analytical column. Three differently sized pDNA molecules of 3.0 kbp, 5.2 kbp and 14.0 kbp were used. Plasmid DNA was injected onto columns under the binding conditions and the separation of the isoforms took place by increasing the ionic strength of the elution buffer. While there was no substantial decrease of the recovered supercoiled and linear isoforms of the pDNA with the increase of the plasmid size and with the increase of the flow rate (recoveries in all cases larger than 75%), a pronounced decrease of the oc isoform recovery was observed. The entrapment of the oc pDNA isoform occurred under non-binding conditions as well. The partial oc isoform elution from the column could be achieved by decreasing the flow rate of the elution mobile phase. The results suggested a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material as well as within the intra-particle space of the non-porous particles. This phenomenon was observed on both types of the stationary phase morphologies and could only be connected to the size of a void space through which the pDNA needs to migrate. A prediction of reversible pDNA entrapment was successfully estimated with the calculation of Peclet numbers, Pe, which defines the ratio between a convective and diffusive mass transport.
M. Limonta, N. Lendero Krajnc, U. Vidic, L. Zumalacárregui
Biochemical Engineering Journal 80 (2013) 14-18
The pIDKE2 plasmid is the main component of the CIGB's candidate vaccine against Hepatitis C virus (HVC), which is being used in HCV chronically-infected individuals during clinical trials phase 1 and 2. The designed downstream process of pIDKE2 plasmid produces up to 179 g/year. In order to conduct further improvements, modelling of the downstream process was performed. A methodology based on process analysis tools, such as experimental design and modelling was established to identify factors with the highest influence on production cost and the amount of annual plasmid. Taking into account that the pIDKE2 downstream process designed is in its initial stages of development, CIM technology was evaluated as a new manufacturing process on lab scale. Purity and recovery of CIM technology was better than porous particle matrix, thus SuperPro Designer was used in order to simulate the purification process. Cost efficiency optimization of the pIDKE2 downstream process was done with the simulation model.
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.
A. Ghanem, R. Healey, F. G. Adly
Analytica Chimica Acta 760 (2013) 1-15
Abstract
Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cellmediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.
A. Romanovskaya, L. P. Sarina, D. H. Bamford, M. M. Poranen
Journal of Chromatography A (2013)
Recent advances in the field of RNA interference and new cost-effective approaches for large-scale double-stranded RNA (dsRNA) synthesis have fuelled the demand for robust high-performance purification techniques suitable for dsRNA molecules of various lengths. To address this issue, we developed an improved dsRNA purification method based on anion exchange chromatography utilizing convective interaction media (CIM) monolithic columns. To evaluate column performance we synthesized a selection of dsRNA molecules (58–1810 bp) in a one-step enzymatic reaction involving bacteriophage T7 DNA-dependent RNA polymerase and phi6 RNA-dependent RNA polymerase. In addition, small interfering RNAs (siRNAs) of 25–27 bp were generated by Dicer digestion of the genomic dsRNA of bacteriophage phi6. We demonstrated that linearly scalable CIM monolithic quaternary amine (QA) columns can be used as a fast and superior alternative to standard purification methods (e.g. LiCl precipitation) to obtain highly pure dsRNA preparations. The impurities following Dicer treatment were quickly and efficiently removed with the QA CIM monolithic column, yielding siRNA molecules of high purity suitable for potential therapeutic applications. Moreover, baseline separation of dsRNA molecules up to 1 kb in non-denaturing conditions was achieved.
2012
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.
T. Koho, T. Mantyla, P. Laurinmaki, L. Huhti, S. J. Butcher, T. Vesikari, M. S. Kulomaa, V. P. Hytonen
Journal of Virological Methods 181 (2012) 6-11
Recombinant expression of the norovirus capsid protein VP1 leads to self-assembly of non-infectious virus-like particles (VLPs), which are recognized as promising vaccine candidates against norovirus infections. To overcome the scalability issues connected to the ultracentrifugation-based purification strategies used in previous studies, an anion exchange-based purification method for norovirus VLPs was developed in this study. The method consists of precipitation by polyethylene glycol (PEG) and a single anion exchange chromatography step for purifying baculovirus-expressed GII.4 norovirus VLPs, which can be performed within one day. High product purity was obtained using chromatography. The purified material also contained fully assembled monodispersed VLPs, which were recognized by human sera containing polyclonal antibodies against norovirus GII.4.
M. Rupar, M. Ravnikar, M. Tušek-Žnidarič, P. Kramberger, L. Glais, I. Gutiérrez-Aguirre
Journal of Chromatography A, 1272 (2013) 33-40(2013) 33-40
Obtaining pure virus suspensions is an essential step in many applications, such as vaccine production, antibody production, sample preparation for procedures requiring enrichment in viruses and other in vitro characterizations. Purification procedures usually consist of complex, long lasting and tedious protocols involving several ultracentrifugation steps. Such complexity is particularly evident in the case of plant viruses, where the virus needs to be isolated from the complex plant tissue matrix. Convective Interaction Media (CIM) monoliths are chromatographic supports that have been successfully utilized for the purification of large bio-molecules such as viruses, virus like particles and plasmids from various matrixes. In this study a CIM monolith based procedure was developed for the fast purification from plant tissue of the filamentous Potato virus Y (PVY) (virion size, 740 nm × 11 nm), which is one of the most important plant viruses causing great economical losses in potato production. Different mobile phases, chemistries and sample preparation strategies were tested. The presence of the virus in the chromatographic fraction was monitored with viral RNA quantification (RT-qPCR), viral protein purity estimation (SDS-PAGE) and viral particle integrity observation (transmission electron microscopy). The optimized procedure involves initial clarification steps, followed by chromatography using CIM quaternary amine (QA) monolithic disk column. In comparison to classical purification procedure involving ultracentrifugation through sucrose and caesium chloride, the developed CIM-QA purification achieved comparable yield, concentration and purity. Plant nucleic acids were successfully removed. Purification showed good reproducibility and moreover it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where a filamentous virus was purified using CIM monolithic supports. The advantages of this new purification procedure make it an attractive method in serological diagnostic tool production, which requires purified viruses for the immunization step. Moreover, the outcome of this study could serve as starting point for the improvement of the purification methods of other important filamentous viruses.
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M. M. Segura, M. Puig, M. Monfar, M. Chillon
HUMAN GENE THERAPY METHODS 23:182–197 (June 2012)
Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ul- tracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarifi- cation and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contami- nating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58– 69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks.
N. Mehle, M. Ravnikar
Water research 46 (2012) 4902 - 4917
The presence of plant viruses outside their plant host or insect vectors has not been studied intensively. This is due, in part, to the lack of effective detection methods that would enable their detection in difficult matrixes and in low titres, and support the search for unknown viruses. Recently, new and sensitive methods for detecting viruses have resulted in a deeper insight into plant virus movement through, and transmission between, plants. In this review, we have focused on plant viruses found in environmental waters and their detection. Infectious plant pathogenic viruses from at least 7 different genera have been found in aqueous environment. The majority of the plant pathogenic viruses so far recovered from environmental waters are very stable, they can infect plants via the roots without the aid of a vector and often have a wide host range. The release of such viruses from plants can lead to their dissemination in streams, lakes, and rivers, thereby ensuring the long-distance spread of viruses that otherwise, under natural conditions, would remain restricted to limited areas.
The possible sources and survival of plant viruses in waters are therefore discussed. Due to the widespread use of hydroponic systems and intensive irrigation in horticulture, the review is focused on the possibility and importance of spreading viral infection by water, together with measures for preventing the spread of viruses. The development of new methods for detecting multiple plant viruses at the same time, like microarrays or new generation sequencing, will facilitate the monitoring of environmental waters and waters used for irrigation and in hydroponic systems. It is reasonable to expect that the list of plant viruses found in waters will thereby be expanded considerably. This will emphasize the need for further studies to determine the biological significance of water-mediated transport.
V. Bandeira, C. Peixoto, A. F. Rodrigues, P. E. Cruz, P. M. Alves, A. S. Coroadinha, M. J. T. Carrondo
Human Gene Therapy Methods 23:1-9 (August 2012)
Lentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturing and purification of these vectors still present major challenges, mainly because of the low stability of the virus, essentially due to the fragility of the membrane envelope. The main goal of this work was the establishment of a fast, scalable, and robust downstream protocol for LVs, combining microfiltration, anion-exchange, and ultrafiltration membrane technologies toward maximization of infectious LVs recovery. CIM® (Convective Interaction Media) monolithic columns with diethylaminoethanol (DEAE) anion exchangers were used for the purification of clarified LV supernatants, allowing infectious vector recoveries of 80%, which is 10% higher than the values currently reported in the literature. These recoveries, combined with the results obtained after optimization of the remaining downstream purification steps, resulted in overall infectious LV yields of 36%. Moreover, the inclusion of a Benzonase step allowed a removal of approximately 99% of DNA impurities. The entire downstream processing strategy herein described was conceived based on disposable and easily scalable technologies. Overall, CIM DEAE columns have shown to be a good alternative for the purification of LVs, since they allow faster processing of the viral bulks and enhanced preservation of virus biological activity, consequently, increasing infectious vector recoveries.
E. M. Adriaenssens et al.
SciVerse ScienceDirect, Virology, 2012
The use of anion-exchange chromatography was investigated as an alternative method to concentrate and purify bacterial viruses, and parameters for different bacteriophages were compared. Chromatography was performed with Convective Interactive Media® monoliths, with three different volumes and two matrix chemistries. Eleven morphologically distinct phages were tested, infecting five different bacterial species. For each of the phages tested, a protocol was optimized, including the choice of column chemistry, loading, buffer and elution conditions. The capacity and recovery of the phages on the columns varied considerably between phages. We conclude that anion-exchange chromatography with monoliths is a valid alternative to the more traditional CsCl purification, has upscaling advantages, but it requires more extensive optimization.
M. Lock, M. R. Alvira, J. M. Wilson
HUMAN GENE THERAPY METHODS: Part B 23:56-64 (February 2012)
Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of everincreasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium.
J. Ruščić, I. Gutierrez-Aguirre, L. Urbas, P. Kramberger, N. Mehle, D. Škorić, M. Barut, M. Ravnikar, M. Krajačić
Journal of Chromatography A, 1274 (2013) 129-136
Potato spindle tuber viroid (PSTVd) is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and unencapsidated RNA molecule with only 356–360 nucleotides and no coding capacity. Because of its peculiar structural features, it is very stable ex vivo and it is easily transmitted mechanically by contaminated hands, tools, machinery, etc. In this work, we describe the development and optimization of a method for concentrating PSTVd using Convective Interaction Media (CIM) monolithic columns. The ion-exchange chromatography on diethylamine (DEAE) monolithic analytical column (CIMac DEAE-0.1 mL) resulted in up to 30% PSTVd recovery whilst the hydrophobic interaction chromatography on C4 monolithic analytical column (CIMac C4-0.1 mL) improved it up to 60%. This was due to the fact that the binding of the viroid to the C4 matrix was less strong than to the highly charged anion-exchange matrix and could be easier and more completely eluted under the applied chromatographic conditions. Based on these preliminary results, a C4 HLD-1 (High Ligand Density) 1 mL monolithic tube column was selected for further experiments. One-litre-water samples were mixed with different viroid quantities and loaded onto the column. By using reverse transcription quantitative polymerase chain reaction (RT-qPCR), the viroid RNA was quantified in the elution fraction (≈5 mL) indicating that 70% of the viroid was recovered and concentrated by at least two orders of magnitude. This approach will be helpful in screening irrigation waters and/or hydroponic systems’ nutrient solutions for the presence of even extremely low concentrations of PSTVd.
2011
F. Smrekar, M. Ciringer, J. Jančar, P. Raspor, A. Štrancar, A. Podgornik
Journal of Separation Science 2011, 34, 2152-2158
A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on-line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method.