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 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.

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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.

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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.

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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.

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A. Martinčič, M. Cemazar, G. Sersa, V. Kovač, R. Milačič, J. Ščančar

Talanta 116 (2013)141-148

Conjoint liquid chromatography (CLC) on monolithic convective interaction media (CIM) disks coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP-MS) detectors was used for the first time in speciation analysis of Pt in human serum spiked with Pt-based chemotherapeutics. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. Such a set-up allows rapid two-dimensional separation by affinity and ion-exchange (IE) modes to be carried out in a single chromatographic run. By applying isocratic elution with Tris–HCl–NaHCO3 buffer (pH 7.4) in the first minute, followed by gradient elution with 1 mol L-1 NH4Cl (pH 7.4) in the next 9 min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Pt from Pt bound to transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Further elution with acetic acid (AcOH) in the next 3 min allowed separation of Pt associated with IgG. Separated Pt species were quantified by post-column isotope dilution—ICP-MS. Pt recovery on the CLC column was close to 100%. In comparison to commonly applied procedures that involve separation of protein peaks by size-exclusion chromatography (SEC) followed by IE separation of metal-based chemotherapeutic fractions bound to serum proteins, the CLC method developed is much faster and simpler. Its sensitivity (LOQs adequate for quantification of all separated Pt species, lower than 2.4 ng Pt mL-1), good selectivity and method repeatability (RSD±3%) enabled investigation of the kinetics of interaction of Pt-based chemotherapeutics with serum proteins and the distribution of Pt species in spiked human serum. Pt species present in spiked serum were bound preferentially to HSA. The proportion of Pt associated with IgG and Tf was lower than 13%. Cisplatin and especially oxaliplatin react rapidly with serum proteins, while carboplatin much less. The method developed may be reliably applied in preclinical and clinical studies of the kinetics of the interaction and distribution of different metallodrugs with proteins in blood serum.

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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.

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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.

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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.

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O. Zitka, S. Skalickova, S. Krizkova, M. Vlkova, V. Adam, R. Kizek

Chromatographia (2013) 76:611-619

In this study, we optimized method for the isolation and detection of lactoferrin from human saliva using 3 mm short monolithic disc. We optimized the conditions for separation as flow rate 4 mL min-1 and ionic strength of effluent as 2 M·NaCl. We estimated limit of detection of whole method, which was hyphenated to the Bradford’s assay, down to 100 ng mL-1. The purity of the isolated fractions was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and recovery of isolation was found to be 51 % using minimally processed sample of saliva. Further, we tested the optimized method on group of healthy volunteers (n = 7). We were able to distinguish between the healthy subjects and subject suffering from celiac disease, which reported at least 2.5× higher level of lactoferrin in comparison to healthy ones. The results were correlated with standard enzyme-linked immunosorbent assay (ELISA) kit with obtained correlation coefficient R2 = 0.8446. Analysis of lactoferrin in saliva by monolithic disc and subsequent offline photometric detection is faster and cheaper method compared to ELISA commercial kit. The total analysis of one sample takes.

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D. A. Ribeiro, D. F. Passos, H. C. Ferraz, L. R. Castilho

Journal of Chromatography B, 938 (2013) 111-118

Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose® FF and Fractogel® TMAE, the monolith CIM® QA and the membrane adsorber Sartobind® Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca2+ ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45 mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.

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J. A. Martin, P. Parekh, Y. Kim, T. E. Morey, K. Sefah, N. Gravenstein, D. M. Dennis, W. Tan

PLOS ONE, March 2013, Volume 8, Issue 3, e57341

Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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