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.
A. Ghanem, R. Healey, F. G. Adly
Analytica Chimica Acta 760 (2013) 1-15
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.
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.
BioProcess International, November 2012, pg. 31-42
Monoclonal antibodies (MAbs) remain the largest segment of the biopharmaceutical market, but they are not the only recombinant proteins in development. Remember that the first biopharmaceutical approved for sale was recombinant insulin — a hormone — back in the 1980s. And proteins aren't the only recombinant biologics. The sector has expanded since then to include gene therapies and viral vectors, vaccines, and even cells and tissues. Companies around the world are developing such products for cancer, neurological, infectious disease, metabolic, autoimmune, and cardiovascular disorders, to name just the most prominent. And although MAbs are finally fulfilling their “magic bullet” promise, many other approaches are becoming available to drug developers targeting those markets — and others.
Meanwhile, funding challenges are increasing emphasis on manufacturing and development efficiencies. Even though total funding of the biotechnology industry has rebounded since the 2008 recession — from about US$13 billion for the United States industry in 2008 to about $21 billion in 2010, for example — a growing share of that money is going to the less risky investments. According to Ernst & Young's 2011 Beyond Borders report, that means mature and already-profitable companies are taking a larger portion of the financial pie.
At the same time, the average number of drug approvals per year has decreased: from about three dozen in the United States from 1996 to 2004 to under two dozen for the years since. And even though markets are opening up in China, India, and other countries, the cost of doing business on a global scale makes it no easy task to reach them. So biopharmaceutical companies need to curb the rise of development and manufacturing costs. Single-use technologies are helping with the latter in large part. And platform technologies have helped antibody makers shorten development times by starting out with certain rules of thumb — rather than trying out hundreds of available purification technologies, for example, in many different combinations to find what works best for every new product candidate.
Do nonantibody makers have similar options when it comes to their own process development work? As is so often the case in bioprocessing, the answer to that question is “It depends. ..” on the product class; on the expression system; and on the regulatory history of the company, process, and type of molecule.
K. Sushma, C. J. Bilgimol, M. A. Vijayalakshmi, P. K. Satheeshkumar
Journal of Chromatography B, 891 - 892 (2012) 90 - 93(2012) 90 - 93
Anti TNF-α molecules are important as therapeutic agents for many of the autoimmune diseases in chronic stage. Here we report the expression and purification of a recombinant single chain variable fragment (ScFv) specific to TNF-α from inclusion bodies. In contrast to the conventional on column refolding using the soft gel supports, an efficient methodology using monolithic matrix has been employed. Nickel (II) coupled to convective interaction media (CIM) support was utilized for this purpose with 6 M guanidine hydrochloride (GuHCl) as the chaotropic agent. The protein purified after solubilization and refolding proved to be biologically active with an IC50.
M. Srajer Gajdosik, J. Clifton, D. Josić,
Journal of Chromatography A, 1239 (2012) 1- 9
Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed.
A. Albreht, I. Vovk
Journal of Chromatography A, 1227 (2012) 210-218
The separation and isolation of major whey proteins is already extensively covered in the literature although no study has been published in which monolithic columns were used. In our research we present, for the first time, the use of short convective interaction media (CIM) monolithic columns for the separation of all major whey proteins and isolation of β-lactoglobulin variant A and B (β-LgA and β-LgB) from a commercial product whey isolate (WI). Although our primary interest was directed towards finding a proper monolithic column and chromatographic conditions for the purification and isolation of β-LgA and β-LgB, three additional analytical LC methods, each having its own potential application target, were also developed in the course of our research. On the monolithic diethylaminoethyl convective interaction media analytical column (CIMac DEAE), the separation of major whey proteins was achieved by gradually lowering the pH of the mobile phase. The ever-so-hard obtainable linear external pH gradient was very linear in the range of pH 5.5–3 and the developed ion-exchange (IE) high-performance liquid chromatographic (HPLC) method was amenable to mass spectrometry (MS). A very fast baseline separation, with UV detection, of all major whey proteins was achieved on a prototype CIMac reversed-phase styrene-divinylbenzene (RP-SDVB) monolithic column in only 4 min and the performance of this column proved superior in comparison with the packed particle POROS perfusion column. The developed RP-HPLC–MS method is fast and, due to the MS detector, can offer low limits of detection and quantitation. Finally, in order to fulfill our primary interest, a scale-up method was developed, using a prototype 8 mL analogue of the CIMac RP-SDVB column, for the isolation of native and chemically unmodified β-LgA and β-LgB from WI with purities higher than 90% and 81%, respectively. The proteins were to be used in further protein–ligand binding studies. The developed methods excel in speed of the analysis, sensitivity, resolution, and simplicity. Thus, it is shown for the first time that short monolithic columns are applicable to the separation and isolation of major whey proteins and that their use has some obvious benefits.
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.
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.
ChemieXtra 3/2012 pp 30-33
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.
E. S. Sinitsyna, J. G. Walter, E. G. Vlakh, F. Stahl, C. Kasper, T. B. Tennikova
Talanta 93 (2012) 139-146
Macroporous monoliths with different surface functionalization (reactive groups) were utilized as platforms for DNA analysis in microarray format. The slides based on a copolymer glycidyl methacrylate-co- ethylene dimethacrylate (GMA-EDMA) have been chosen as well known and thoroughly studied standard. In particular, this material has been used at optimization of DNA microanalytical procedure.
The concentration and pH of spotting solution, immobilization temperature and time, blocking agent and coupling reaction duration were selected as varied parameters. The efficiency of analysis performed on 3-D monolithic platforms was compared to that established for commercially available glass slides. As a practical example, a diagnostic test for detection of CFTR gene mutation was carried out. Additionally, the part of presented work was devoted to preparation of aptamer-based test-system that allowed successful and highly sensitive detection both of DNA and protein.