Publications
2012
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.
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.
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.
M. Pucic, A. Knezevic, J. Vidic, B. Adamczyk, M. Novokmet, O. Polasek, O. Gornik, S. Supraha-Goreta, M. R. Wormald, I. Redzic, H. Campbell, A. Wright, N. D. Hastie, J. F. Wilson, I. Rudan, M. Wuhrer, P. M. Rudd, Dj. Josic, and G. Lauc
Mol Cell Proteomics. Oct 2011; published online Jun 8, 2011
All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously.
Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.
Z. Jiang, N. W. Smith, Z. Liu
Journal of Chromatography A, 1218 (2011) 2350-2361
Hydrophilic interaction chromatography (HILIC) has experienced increasing attention in recent years. Much research has been carried out in the area of HILIC separation mechanisms, column techniques and applications. Because of their good permeability, low resistance to mass transfer and easy preparation within capillaries, hydrophilic monolithic columns represent a trend among novel HILIC column techniques. This review attempts to present an overview of the preparation and applications of HILIC monolithic columns carried out in the past decade. The separation mechanism of various hydrophilic monolithic stationary phases is also reviewed.
M. R. Etzel, T. Bund
Journal of Chromatography A, 1218 (2011) 2445-2450
Proteins conjugated to neutral biopolymers are of keen interest to the food and pharmaceutical industries. Conjugated proteins are larger and more charge shielded than un-reacted proteins, making purification difficult using conventional beaded chromatographic supports because of slow mass transfer rates, weak binding, and viscous solutions. Past methods developed for pharmaceuticals are unsuitable for foods. In this work, a food-grade whey protein–dextran conjugate was purified from a feed solution also containing un-reacted protein and dextran using either a column packed with 800 mL of a beaded support that was specifically designed for purification of conjugated proteins or an 8 mL tube monolith. The monolith gave a similar dynamic binding capacity as the beaded support (4–6 g/L), at a 42-fold greater mass productivity, and 48-fold higher flow rate, albeit at somewhat lower conjugate purity. Performance of the monolith did not depend on flow rate. In conclusion, monoliths were found to be well suited for the purification of whey protein–dextran conjugates.
I. Pulko, V. Smrekar, A. Podgornik, P. Krajnc
Journal of Chromatography A, 1218 (2011) 2396-2401
Approximately 25 cm × 25 cm large sheets of crosslinked highly porous poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate-co-ethylhexyl methacrylate) membranes with an average thicknesses between 285 and 565 μm were prepared by casting a high internal phase emulsion (HIPE) containing monomers onto glass substrates and subsequent polymerisation. Open cellular porous polyHIPE type membranes were obtained with large pores (cavity) sizes between 3 and 10 μm while interconnecting pores were between 1 and 3 μm. The percentage of ethylhexyl acrylate and ethyleneglycol dimethacrylate influenced the flexibility and morphology of the resulting membranes. Porous membranes were chemically modified with diethylamine to yield functionalised supports for ion exchange chromatography. Cylindrical housings were used for positioning of the membranes and allowing flow of the mobile phase. Pulse experiments were used to study the flow characteristics and a homogeneous flow through the entire area of the membrane was found. Bovine serum albumin was purified by a 8 ml column containing functional membrane in modular shape; dynamic binding capacity was measured to be as high as 45 mg/ml.
C. Valasek, J. Cole, F. Hensel, P. Ye, M. A. Conner, M. E. Ultee
BioProcess International, Vol. 9, No. 11, December 2011, pp. 28–37
Immunoglobulin G (IgG) antibodies have been used to treat cancer for many years (1). Another class of antibodies—immunoglobulin M (IgM)—has been overlooked in spite of offering unique advantages that make them highly desirable as cancer therapeutics. Serving a valuable function in our innate immune system, IgM antibodies are the first to be secreted when an abnormal cell is present (2). These antibodies play a critical role in recognition and elimination of infectious particles (3,4), in removal of intracellular components, and in immunosurveillance mechanisms against malignant cells (5,6). IgMs also can bind to multiple copies of a target on a cancer cell surface. Such high avidity leads to cross-linking and more effective cell killing (7).
P. Gagnon, F. Hensel, S. Lee, S. Zaidi
Journal of Chromatography A, 1218 (2011) 2405-2412
This study documents the presence of stable complexes between monoclonal IgM and genomic DNA in freshly harvested mammalian cell culture supernatants. 75% of the complex population elutes from size exclusion chromatography with the same retention volume as IgM. DNA comprises 24% of the complex mass, corresponding to an average of 347 base pairs per IgM molecule, distributed among fragments smaller than about 115 base pairs. Electrostatic interactions appear to provide most of the binding energy, with secondary stabilization by hydrogen bonding and metal affinity. DNA-dominant complexes are unretained by bioaffinity chromatography, while IgM-dominant complexes are retained and coelute with IgM. DNA-dominant complexes are repelled from cation exchangers, while IgM-dominant complexes are retained and partially dissociated. Partially dissociated forms elute in order of decreasing DNA content. The same pattern is observed with hydrophobic interaction chromatography. All complex compositions bind to anion exchangers and elute in order of increasing DNA content. A porous particle anion exchanger was unable to dissociate DNA from IgM. Monolithic anion exchangers, offering up to15-fold higher charge density, achieved nearly complete complex dissociation. The charge-dense monolith surface appears to outcompete IgM for the DNA. Monoliths also exhibit more than double the IgM dynamic binding capacity of the porous particle anion exchanger, apparently due to better surface accessibility and more efficient mass transfer.
P. Gagnon, G. Rodriquez, S. Zaidi
Journal of Chromatography A, 1218 (2011) 2402-2404
A basic method for dissociation and fractionation of monoclonal IgG heavy and light chain is described. It employs less noxious and hazardous reagents than the classical mercaptoethanol/propionic acid process and replaces size exclusion chromatography with cation exchange on a monolith to improve productivity. Significant scope remains to refine the conditions. The method can be applied to other disulfide bonded proteins with significant affinity for cation exchangers.
S. Neff, A. Jungbauer
Journal of Chromatography A, 1218 (2011) 2374-2380
We have developed a method for quantification of a specific monoclonal IgM directed toward embryonic stem cells based on a peptide affinity monolith. A peptide affinity ligand with the sequence C–C–H–Q–R–L–S–Q–R–K was obtained by epitope mapping using peptide SPOT synthesis. The peptide ligand was covalently immobilized by coupling the N-terminal cysteine to a monolithic disk that was previously modified with iodated spacer molecules. The monolithic disc was used for quantification of purified IgM and for IgM present in mammalian cell culture supernatant. We observed 17% unspecific binding of IgM to the monolithic disk and additionally a product loss in the flow through of 20%. Nevertheless, calibration curves had high correlation coefficients and inter/intra-assay variability experiments proved sufficient precision of the method. A limit of quantification of 51.69 μg/mL for purified IgM and 48.40 μg/mL for IgM in cell culture supernatant could be calculated. The binding capacity was consistent within the period of the study which included more than 200 cycles. The analysis time of less than 2 min is an advantage over existing chromatographic methods that rely on pore diffusion.
A. Mönster, O. Hiller, D. Grüger, R. Blasczyk, C. Kasper
Journal of Chromatography A, 1218 (2011) 706–710
Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM®) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM® Disk with epoxy chemistry. After this, the immobilized CIM® Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM® Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap® metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.
R. D. Arrua, C. I. Alvarez Igarzabal
J. Sep. Sci. 2011, 34, 1974–1987
In the early 1990s, three research groups simultaneously developed continuous macroporous rod-shaped polymeric systems to eliminate the problem of flow through the interparticle spaces generally presented by the chromatography columns that use particles as filler. The great advantage of those materials, forming a continuous phase rod, is to increase the mass transfer by convective transport, as the mobile phase is forced to go through all means of separation, in contrast to particulate media where the mobile phase flows through the interparticle spaces. Due to their special characteristics, the monolithic polymers are used as base-supports in different separation techniques, those chromatographic processes being the most important and, to a greater extent, those involving the separation of biomolecules as in the case of affinity chromatography. This mini-review reports the contributions of several groups to the development of macroporous monoliths and their modification by immobilization of specific ligands on the products for their application in affinity chromatography.
S. Neff, A. Jungbauer
Journal of Chromatography A, 1218 (2011) 2374–2380
We have developed a method for quantification of a specific monoclonal IgM directed toward embryonic stem cells based on a peptide affinity monolith. A peptide affinity ligand with the sequence C–C–H–Q–R–L–S–Q–R–K was obtained by epitope mapping using peptide SPOT synthesis. The peptide ligand was covalently immobilized by coupling the N-terminal cysteine to a monolithic disk that was previously modified with iodated spacer molecules. The monolithic disc was used for quantification of purified IgM and for IgM present in mammalian cell culture supernatant. We observed 17% unspecific binding of IgM to the monolithic disk and additionally a product loss in the flow through of 20%. Nevertheless, calibration curves had high correlation coefficients and inter/intra-assay variability experiments proved sufficient precision of the method. A limit of quantification of 51.69 μg/mL for purified IgM and 48.40 μg/mL for IgM in cell culture supernatant could be calculated. The binding capacity was consistent within the period of the study which included more than 200 cycles. The analysis time of less than 2 min is an advantage over existing chromatographic methods that rely on pore diffusion.
H. Shirataki, C. Sudoh, T. Eshima, Y. Yokoyama, K. Okuyama
Journal of Chromatography A, 1218 (2011) 2381–2388
It is widely recognized that membrane adsorbers are powerful tools for the purification of biopharmaceutical protein products and for this reason a novel hollow-fiber AEX type membrane adsorber has been developed. The membrane is characterized by grafted chains including DEA ligands affixed to the pore surfaces of the membrane. In order to estimate the membrane performance, (1) dynamic binding capacities for pure BSA and DNA over a range of solution conductivity and pH, (2) virus reduction by flow-through process, and (3) HCP and DNA removal from cell culture, are evaluated and compared with several other anion-exchange membranes. The novel hollow-fiber membrane is tolerant of high salt concentration when adsorbing BSA and DNA. When challenged with a solution containing IgG the membrane has high impurity removal further indicating this hollow-fiber based membrane adsorber is an effective tool for purification of biopharmaceutical protein products including IgG.
M. J. Shin, L. Tan, M. H. Jeong, J.-H. Kim, W.-S. Choe
Journal of Chromatography A, 1218 (2011) 5273-5278
Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl2-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification.
2010
P. Gagnon
BioProcess International, Nov 2010
The enabling value of monoliths was strongly in evidence at the 4th International Monolith Symposium, held 29 May – 2 June in the Adriatic resort city of Portoroz, Slovenia. Forty-seven oral presentations and 34 posters highlighted important advances in vaccines, gene therapy, phage therapy for infectious disease, and monoclonal antibodies, as well as continuing advances in the performance of monoliths themselves. As these fields advance in parallel, it becomes increasingly apparent that monoliths offer industrial capabilities substantially beyond traditional methods.