Publications
2013
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.
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
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.
R. Milačič, D. Ajlec, T. Zuliani, D. Žigon, J. Ščančar
Talanta 101 (2012) 203-210
In human milk zinc (Zn) is bound to proteins and low molecular mass (LMM) ligands. Numerous investigations demonstrated that Zn bioavailability in human milk is for infant much higher than in cow's milk. It was presumed that in the LMM human milk fraction highly bioavailable Zn-citrate prevails. However, literature data are controversial regarding the amount of Zn-citrate in human milk since analytical procedures reported were not quantitative. So, complex investigation was carried out to develop analytical method for quantitative determination of this biologically important molecule. Studies were performed within the pH range 5–7 by the use of synthetic solutions of Zn-citrate prepared in HEPES, MOPS and MES buffers. Zn-citrate was separated on weak anion-exchange convective interaction media (CIM) diethylaminoethyl (DEAE) monolithic chromatographic column using NH4NO3 as an eluent. Separated Zn species were determined by flame atomic absorption spectrometry (FAAS) or inductively coupled plasma mass spectrometry (ICP-MS). Quantitative separation of Zn-citrate complexes ([Zn(Cit)]- and [Zn(Cit)2]4-; column recoveries 94–102%) and good repeatability and reproducibility of results with relative standard deviation (RSD±3.0%) were obtained. In fractions under the chromatographic peaks Zn-binding ligand was identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS). Limits of detection (LOD) for determination of Zn-citrate species by CIM DEAE-FAAS and CIM DEAE-ICP-MS were 0.01 μg Zn mL-1 and 0.0005 μg Zn mL-1, respectively. Both techniques were sensitive enough for quantification of Zn-citrate in human milk. Results demonstrated that about 23% of total Zn was present in the LMM milk fraction and that LMM-Zn corresponded to Zn-citrate. The developed speciation method represents a reliable analytical tool for investigation of the percentage and the amount of Zn-citrate in human milk.
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
S. H. Lubbad, M. R. Buchmeiser
Journal of Chromatography A, 1218 (2011) 2362-2367
Ring-opening metathesis polymerization- (ROMP) derived monoliths were prepared from 5-norborn-2-enemethyl bromide (NBE-CH2Br) and tris(5-norborn-2-enemethoxy)methylsilane ((NBE-CH2O)3SiCH3) within the confines of surface-silanized borosilicate columns (100 × 3 mm I.D.), applying Grubbs’ first generation benzylidene-type catalyst [RuCl2(PCy3)2(CHPh)]. Monoliths were converted into weak anion exchangers via reaction with diethyl amine. The resulting monolithic anion exchangers demonstrated a very good potential for the anion-exchange separation of nucleic acids applying a phosphate buffer (0.05 mol/L, pH 7) and NaCl (1.0 mol/L) as a gradient former. Fast and efficient separations, indicated by sharp and highly symmetric analyte peaks, were established. Except for the 267 and 298 base pair fragments, the eleven fragments of a ds-pUC18 DNA Hae III digest were baseline separated within ∼8 min. Nineteen fragments of a ds-pBR322 Hae III digest were separated within ∼12 min. There, only the 192 and 213 base pair fragments and the 458, 504 and 540 base pair fragments coeluted. A ds-pUC18 DNA Hae III digest was used as a control analyte in evaluating the influence of organic additives on the mobile phase such as methanol and acetonitrile on nucleic acid separation. Methanol, and even better, acetonitrile improved the separation efficiency and shortened the analysis time.
S. Yamamoto, T. Okada, M. Abe, N. Yoshimoto
Journal of Chromatography A, 1218 (2011) 2460-2466
The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3 mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-μm porous bead IEC column (Resource Q, 6.4 mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position KR. HETP values were constant (ca. 0.003–0.005 cm) when KR > 5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50–100 plates for the 3 mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume = 0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.