Publications
2023
Lucija Rebula, Andrej Raspor, Mojca Bavčar, Aleš Štrancar and Maja Leskovec
Journal of Chromatography B, Volume 1217, 15 February 2023
Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.
In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.
2020
Hietala V, Horsma-Heikkinen J, Carron A, Skurnik M, Kiljunen S.
Frontiers in microbiology vol. 10 1674. 23 Jul. 2019
Abstract
The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiM_fHyAci03, and Staphylococcus phage vB_SauM_fRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_fHoEco02 lysate from 3.5 × 104 Endotoxin Units (EU)/109 plaque forming units (PFU) to 0.09 EU/109 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/109 PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/109 PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.
Keywords: antibiotic resistance, bacteriophage, phage therapy, endotoxin, enterotoxin
2019
Calef Sánchez-Trasviña, Marco Rito-Palomares, and José González-Valdez
Advances in Polymer Technology, Volume 2019, December 12 2019, 10 pages
Abstract
PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.
2015
J-P Pirnay et al.
Pharm Res, Springer, 14 Jan 2015
The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge.
To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies.
This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.
2014
F. W. Krainer, R. Pletzenauer, L. Rossetti, C. Herwig, A. Glieder, O. Spadiut
Protein Expression and Purification 95 (2014) 104–112
The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.
F. W. Krainer, R. Pletzenauer, L. Rossetti, C. Herwig, A. Glieder, O. Spadiut
Protein Expression and Purification 95 (2014) 104–112
The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.
2013
M. Bartolini, I. W. Wainer, C. Bertucci, V. Andrisano
Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 77-81
Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2 mm × 6 mm i.d.), under a three-solvent gradient elution mode and UV detection at 256 nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.
F. Ibrahim, C. Andre, R. Aljhni, T. Gharbi, Y. C. Guillaume
Journal of Molecular Catalysis B: Enzymatic 94 (2013) 136-140
Acetylcholinesterase (AChE) is a serine protease that hydrolyzes the neurotransmitter acetylcholine. Here, the effects of hydroxyl radical (OH•) and nitric oxide (NO) on AChE activity were studied using a biochromatographic process. The enzyme was immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk. The AChE enzymatic mechanism was demonstrated from the chromatographic peak shape. A decrease in AChE activity was observed for each concentration of NO, while OH• dot radical formation led to an increase in the rate of enzymatic catalysis. Michaelis–Menten and Lineweaver–Burk plots were obtained in the presence or absence of the free radicals and their effects on Km and Vmax were evaluated. Our results indicated classical deactivation/activation kinetics without significant influence on the rate of substrate binding. The variation in transition state energies (ΔΔGES) induced by the free radicals indicated that a conformational change was occurring in the active site, while changes in the binding site were negligible. These results clearly demonstrate the direct role of OH• dot and NO on AChE activity and confirm the role they may play in Alzheimer's disease.
H. G. Schwelberger, J. Feurle, F. Ahrens
Journal of Neural Transmission 120 (2013) 983-986
Diamine oxidase (DAO) was purified to homogeneity from human seminal plasma by consecutive chromatographic fractionation on heparin-sepharose, phenyl-sepharose, CIM-QA, and Superdex 200. Human seminal plasma DAO behaves electrophoretically similar to DAO proteins from other human tissues and has very similar enzymatic properties with histamine and aliphatic diamines being the preferred substrates as well as significant conversion of polyamines. The cellular source and functional importance of DAO in human semen remain to be determined.
E. A. Ponomareva, M. V. Volokitina, D. O. Vinokhodov, E. G. Vlakh, T. B. Tennikova
Anal Bioanal Chem (2013) 405:2195–2206
Immobilized enzyme reactors (IMERs) produced by the covalent attachment of ribonuclease A to macroporous
methacrylate-based monolithic supports using different experimental approaches are discussed and compared. Enzyme immobilization was carried out by direct covalent binding, as well as through attachment via a polymer spacer. The kinetic properties of an IMER operating in either recirculation mode or zonal elution mode were studied. Additionally, the effect of flow rate on the bioconversion efficiency of each IMER sample was examined.
M. V. Volokitina, E. G. Vlakh, G. A. Platonova, D. O. Vinokhodov, T. B. Tennikova
J. Sep. Sci. 2013, 36, 2793-2805
Two ribonuclease A bioreactors based on lab-made macroporous monolithic columns and intended for polynucleotide degradation were prepared using in situ free-radical polymerization. Different methods of enzyme immobilization were applied. In the first case, the biocatalyst molecule was attached to the solid surface via direct covalent binding, while in the second bioreactor the flexible-chain synthetic polymer was used as an intermediate spacer. The effect of temperature, substrate flow rate, and loaded sample volume on the biocatalytic efficiency of the immobilized enzyme was examined. The kinetic parameters of the enzymatic degradation of synthetic polycytidylic acid were calculated and compared to those found for hydrolysis with soluble ribonuclease A. The monitoring of substrate splitting was carried out by means of fast anion-exchange HPLC on an ultra-short monolithic column (disk) using off- and on-line analytical approaches.
2012
J. Subotič, K. Koruza, B. Gabor, M. Peterka, M. Barut, J. Kos, J. Brzin
Affinity Chromatography, Dr. Sameh Magdeldin (Ed.), ISBN: 978-953-51-0325-7, InTech
Proteolytic enzymes (also known as proteases, proteinases or peptidases) offer a wide range of applications. They are routinely used in detergent, leather, food and pharmaceutical industries, as well as in medical and basic research. Therefore, effective isolation procedures are of great importance. The chapter describes the use of recently discovered protease inhibitors from basidiomycetes as affinity chromatography ligands for isolating proteases. Affinity columns with serine and cysteine protease inhibitors immobilized to the natural polymer Sepharose have been prepared, the chromatography procedure optimized and used for isolating proteases from various bacterial, plant and animal sources. The cysteine protease inhibitor macrocypin showed superior characteristics as a ligand, so was selected for immobilization to CIM (Convective Interaction Media) monolithic disks. Different immobilization chemistries and process conditions were optimized to determine the best conditions for high capacity and selectivity. A very effective method for isolating cysteine proteases was developed using affinity chromatography with the fungal cysteine protease inhibitor macrocypin immobilized to a CIM monolithic disk.
2010
F. Mancini, V. Andrisano
Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 355-361
A novel liquid chromatographic method has been developed for use in throughput screening of new inhibitors of human recombinant β-amyloid precursor protein cleaving enzyme (hrBACE1). The approach is based on the use of an immobilized enzyme reactor (IMER) containing the target enzyme (hrBACE1–IMER) and uses fluorescence detection. The bioreactor was prepared by immobilizing hrBACE1 on an ethylendiamine (EDA) monolithic disk (CIM) and a fluorogenic peptide (M-2420) containing the β-secretase site of the Swedish mutation of amyloid precursor protein (APP) was used as substrate. After injection into the hrBACE1–IMER system, M-2420 was enzymatically cleaved, giving rise to a fluorescent methoxycoumaryl-fragment (Rt = 1.6 min), which was separated from the substrate and selectively detected at λexc = 320 and λem = 420 nm. Product and substrate were characterized by using a post monolithic C18 stationary phase coupled to an ion trap mass analyser. A calibration curve was constructed to determine the immobilized hrBACE1–IMER rate of catalysis and kinetic constants. Specificity of the enzymatic cleavage was confirmed by injecting the substrate on a blank CIM-EDA.
The proposed method was validated by the determination of the inhibitory potency of five reference compounds with activities ranked over four order of magnitude (four peptidic inhibitors and a green tea polyphenol, (−)gallocatechin gallate). The obtained results were found in agreement with the data reported in literature, confirming the validity and the applicability of the hrBACE1–IMER as a tool for the fast screening of unknown inhibitors (more than 6 compounds per hour). Moreover, the hrBACE1–IMER showed high stability during the analysis, permitting its use for more than three months without affecting enzyme activity.
E.A. Ponomareva, V.E. Kartuzova, E.G. Vlakh, T.B. Tennikova
Journal of Chromatography B, 878 (2010) 567–574
The effect of different modes of α-chymotrypsin attachment to the surface of methacrylate-based ultrashort monolithic minicolumns on enzyme activity has been studied. The immobilization of protease was carried out via direct covalent binding of chymotrypsin, as well as via its attachment through small and polymer spacers. It was established that the lowest enzyme activity against N-benzoyl-l-tyrosine ethyl ester was found for bioreactor obtained via direct attachment of chymotrypsin to the surface of GMA–EDMA minidisks, whereas the highest parameter close to that determined for dissolved enzyme was found in the case of bioreactor prepared by the introduction of copolymer of 2-deoxy-N-methacryloylamido-d-glucose with N-vinylpyrrolidone and acrolein as a long and flexible polymer spacer. Additionally, the effect of flow rate of substrate recirculation on bioconversion efficiency was examined. Independently on immobilization method, the increase of flow rate led to the raise of biocatalytic efficiency.
2009
C. Delattre, M. A. Vijayalakshmi
Journal of Molecular Catalysis B: Enzymatic 60 (2009) 97–105
Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.
2008
C. Delattre, P. Michaud, M. A. Vijayalakshmi
Journal of Chromatography B, 861 (2008) 203–208
Fast production and purification of α-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 °C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.
2007
K. Isobe, Y. Kawakami
Journal of Chromatography A, 1144 (2007) 85-89(2007) 85-89
A convection interaction media (trade name CIM, Sartorius BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.
I.Vovk, B. Simonovska
Journal of Chromatography B, 849 (2007) 337-343
The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; Mr 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with Mr 82 kDa) and a basic protein with Mr 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM®) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2 M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5 M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with Mr 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH3CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5 mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.
I. Vovk, B. Simonovska
Journal of Chromatography A, 1144 (2007) 90-96(2007) 90-96
An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, Sartorius BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 μg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 μg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5 mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.
M. Benčina, J. Babič, A. Podgornik
Journal of Chromatography A, 1144 (2007) 135–142
In gene therapy and DNA vaccination, RNA removal from DNA preparations is vital and is typically achieved by the addition of ribonuclease into the sample. Removal of ribonuclease from DNA samples requires an additional purification step. An alternative is the implementation of immobilized ribonuclease. In our work, ribonuclease was covalently coupled onto the surface of methacrylate monoliths via epoxy or imidazole carbamate groups. Various immobilization conditions were tested by changing immobilization pH. Ribonuclease immobilized on the monolith via imidazole carbamate groups at pH 9 was found to be six times more active than the ribonuclease immobilized on the monolith via epoxy groups. Under optimal immobilization conditions the Michaelis–Menten constant, Km, for cytidine-2,3-cyclic monophosphate, and turnover number, k3 were 0.52 mM and 4.6 s-1, respectively, and mirrored properties of free enzyme. Enzyme reactor was found to efficiently eliminate RNA contaminants from DNA samples. It was active for several weeks of operation and processed 300 column volumes of sample. Required residence time to eliminate RNA was estimated to be around 0.5 min enabling flow rates above 1 column volume per min.