Publications
2011
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.
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. Trauner, M. H. Bennett, H. D. Williams
PLoS ONE 6(2): e16273. doi:10.1371/ journal.pone.0016273
We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology.
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
M. Abe, P. Akbarzaderaleh, M. Hamachi, N. Yoshimoto, S.Yamamoto
Biotechnol. J. 2010, 5, 477-483
The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEG-ylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.
R. Nian, D. S. Kim, T. Nguyen, L. Tan, C.-W. Kim, I.-K. Yoo, W. S. Choe
Journal of Chromatography A, 1217 (2010) 5910-5949
Toxic heavy metal pollution is a global problem occurring in air, soil as well as water. There is a need for a more cost effective, renewable remediation technique, but most importantly, for a recovery method that is selective for one specific metal of concern. Phage display technology has been used as a powerful tool in the discovery of peptides capable of exhibiting specific affinity to various metals or metal ions. However, traditional phage display is mainly conducted in batch mode, resulting in only one equilibrium state hence low-efficiency selection. It is also unable to monitor the selection process in real time mode. In this study, phage display technique was incorporated with chromatography procedure with the use of a monolithic column, facilitating multiple phage-binding equilibrium states and online monitoring of the selection process in search of affinity peptides to Pb2+. In total, 17 candidate peptides were found and their specificity toward Pb2+ was further investigated with bead-based enzyme immunoassay (EIA). A highly specific Pb2+ binding peptide ThrAsnThrLeuSerAsnAsn (TNTLSNN) was obtained. Based on our knowledge, this is the first report on a new chromatographic biopanning method coupled with monolithic column for the selection of metal ion specific binding peptides. It is expected that this monolith-based chromatographic biopanning will provide a promising approach for a high throughput screening of affinity peptides cognitive of a wide range of target species.
R. R. Prasanna, M. A. Vijayalakshmi
Journal of Chromatography A, 1217 (2010) 3660–3667
Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu2+, Ni2+ and Zn2+ were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu2+ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu2+ and 7–9 mg/ml with Ni2+ for human IgG with MMA buffer. In the case of CIM-IDA-Zn2+ column, the binding capacity was only about 0.5 mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.
A. Dhivya, B. Kumar, R. Prasanna, N. Vijayalakshmi
Chromatographia 2010, 72, December (No. 11/12), pg 1183-1188
Purified monoclonal antibodies (mAb) have been used in therapeutics and some analytical procedures. Purification of mAb by use of high-throughput anion-exchange methacrylate monolithic systems has been attempted in this work. Monolithic macroporous convective interaction media (CIM) with diethylaminoethyl (DEAE) and ethylene diamine (EDA) as anion-exchange ligands were used and evaluated for purification of anti-glycophorin-A IgG1 mouse mAbs from cell culture supernatant (CCS) after precipitation with 50% ammonium sulfate. The adsorption and elution of mAb from the CCS on CIM-DEAE and CIM-EDA disks were studied with three different buffer systems, acetate, MOPS (3-(N-morpholino)propanesulfonic acid), and Tris, to study the effect of the nature of buffer ions and to find the optimum buffer conditions for purification of mAb. The optimum buffers for purification of mAb using CIM-DEAE and CIM-EDA were 50 mM acetate buffer, pH 5.1 and 20 mM Tris buffer, pH 8.0, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) showed the antibody fractions obtained were highly pure, with high antigen-binding efficiency. High specific activity with purification factors of 130 ± 34 (unretained fraction with acetate buffer) and 74 ± 13 (fraction eluted with Tris buffer containing 0.6 M NaCl) was obtained for IgG1 using the CIM-DEAE and CIM-EDA disks, respectively. The results indicate that rapid separation and efficient recovery of high-purity anti-glycophorin-A mAbs could be achieved by use of anion-exchange CIM disks.
F. Mancini, V. Andrisano
Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 355-361
A novel liquid chromatographic method has been developed for use in throughput screening of new inhibitors of human recombinant β-amyloid precursor protein cleaving enzyme (hrBACE1). The approach is based on the use of an immobilized enzyme reactor (IMER) containing the target enzyme (hrBACE1–IMER) and uses fluorescence detection. The bioreactor was prepared by immobilizing hrBACE1 on an ethylendiamine (EDA) monolithic disk (CIM) and a fluorogenic peptide (M-2420) containing the β-secretase site of the Swedish mutation of amyloid precursor protein (APP) was used as substrate. After injection into the hrBACE1–IMER system, M-2420 was enzymatically cleaved, giving rise to a fluorescent methoxycoumaryl-fragment (Rt = 1.6 min), which was separated from the substrate and selectively detected at λexc = 320 and λem = 420 nm. Product and substrate were characterized by using a post monolithic C18 stationary phase coupled to an ion trap mass analyser. A calibration curve was constructed to determine the immobilized hrBACE1–IMER rate of catalysis and kinetic constants. Specificity of the enzymatic cleavage was confirmed by injecting the substrate on a blank CIM-EDA.
The proposed method was validated by the determination of the inhibitory potency of five reference compounds with activities ranked over four order of magnitude (four peptidic inhibitors and a green tea polyphenol, (−)gallocatechin gallate). The obtained results were found in agreement with the data reported in literature, confirming the validity and the applicability of the hrBACE1–IMER as a tool for the fast screening of unknown inhibitors (more than 6 compounds per hour). Moreover, the hrBACE1–IMER showed high stability during the analysis, permitting its use for more than three months without affecting enzyme activity.
F. Smrekar, A. Podgornik, M. Ciringer, S. Kontrec, P. Raspor, A. Štrancar, M. Peterka
Vaccine 28 (2010) 2039–2045
Plasmid DNA (pDNA) used in vaccination and gene therapy has to be highly pure and homogenous, which point out necessity to develop efficient, reproducible and scalable downstream process. Convective Interaction Media (CIM) monolithic chromatographic supports being designed for purification of large molecules and nanoparticles seem to be a matrix of choice for pDNA purification. In present work we describe a pDNA purification process designed on two different CIM monolithic columns, based on anion-exchange (AEX) chromatography and hydrophobic interaction chromatography (HIC) chemistry. HIC monolith enabled separation of supercoiled (sc) pDNA from open circular (oc) pDNA, genomic DNA (gDNA) and endotoxins regardless to flow rates in the range at least up to 380 cm/h. Dynamic binding capacity of new HIC monolith is up to 4 mg of pDNA per milliliter of support. Combination of both chromatographic steps using optimized CaCl2 precipitation enabled production of pure pDNA, satisfying all regulatory requirements. Process was found to be reproducible, scalable, and exhibits high productivity. In addition, in-line monitoring of pDNA purification process is shown, using CIM DEAE disk monolithic columns.
N. Lendero Krajnc, F. Smrekar, A. Štrancar, A. Podgornik
Journal of Chromatography A, 1218 (2011) 2413-2424
The objective of this study was to investigate the behavior of large plasmids on the monolithic columns under binding and nonbinding conditions. The pressure drop measurements under nonbinding conditions demonstrated that the flow velocities under which plasmid passing monolith became hindered by the monolithic pore structure depended on the plasmid size as well as on the average monolith pore size; however, they were all very high exceeding the values encountered when applying CIM monolithic columns at their maximal flow rate. The impact of the ligand density and the salt concentration in loading buffer on binding capacity of the monolith for different sized plasmids was examined. For all plasmids the increase of dynamic binding capacity with the increase of salt concentration in the loading solution was observed reaching maximum of 7.1 mg/mL at 0.4 M NaCl for 21 kbp, 12.0 mg/mL at 0.4 M NaCl for 39.4 kbp and 8.4 mg/mL at 0.5 M NaCl for 62.1 kbp. Analysis of the pressure drop data measured on the monolithic column during plasmid loading revealed different patterns of plasmid binding to the surface, showing “car-parking problem” phenomena under certain conditions. In addition, layer thickness of adsorbed plasmid was estimated and at maximal dynamic binding capacity it matched calculated plasmid radius of gyration. Finally, it was found that the adsorbed plasmid layer acts similarly as the grafted layer responding to changes in solution's ionic strength as well as mobile phase flow rate and that the density of plasmid layer depends on the plasmid size and also loading conditions.
M. Peterka, P. Kramberger, A. Štrancar
Wang, Perry G. (ur.). Monolithic chromatography and its modern applications. St Albans: ILM publications, 2010, pg. 489-508
Downstream processing (DSP) for purification can become a significant bottleneck in the production of novel biotherapeutics, such as viral vectors and vaccines (viral or DNA). Although different techniques can be used for the purification of large molecules and particles, liquid chromatography is the preferred method as it achieves the purity required by regulatory agencies. Despite the popularity of conventional chromatographic media, the diffusional mass transfer of large molecules and relatively small pore size remain limiting factors for the efficient separation of large biomolecules and particles.
A. Čevdek, M. Franko
Analytical and Bioanalytical Chemistry 398 (2010), 555-562
This work presents a comparison of convective interaction media (CIM) and controlled pore glass (CPG) as solid supports for immunoglobulin antibodies used in bioanalytical detection of allergens in foodstuffs. A flow-injection manifold with highly sensitive thermal lens spectrometric detection was used for this purpose. Using beta-lactoglobulin, a milk allergen, as a model analyte, CIM disc supports had a higher linear range (0.2–3.5 μg L-1), better reproducibility (intra-day RSD = 1%, inter-day RSD = 10%), lower consumption of reagents, and better immunocolumn stability (1 month, over 240 injections of substrate), while providing comparable LODs (0.1 μg L-1). Application of CIM discs as solid supports in immunocolumns for allergen detection enables fast and sensitive screening of allergens in foodstuffs with sample throughput of up to eight samples per hour.
2009
P. Brne, Y.-P. Lim, A. Podgornik, M. Barut, B. Pihlar, A. Štrancar
Journal of Chromatography A, 1216 (2009) 2658-2663
Convective interaction media (CIM; Sartorius BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.
J. Krenkova, A. Gargano, N. A. Lacher, J. M. Schneiderheinze, F. Švec
Journal of Chromatography A, 1216 (2009) 6824–6830
Poly(glycidyl methacrylate-co-ethylene methacrylate) monoliths have been prepared in 100 μm i.d. capillaries and their epoxy groups hydrolyzed to obtain poly(2,3-dihydroxypropyl methacrylate-co-ethylene methacrylate) matrix. These polymers were then photografted in a single step with 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylic acid to afford stationary phases for a strong and a weak cation exchange chromatography, respectively. Alternatively, poly(ethylene glycol) methacrylate was used for grafting in the first step in order to enhance hydrophilicity of the support followed by photografting with 2-acrylamido-2-methyl-1-propanesulfonic acid or acrylic acid in the second step. These new columns were used for the separation of proteins and peptides. A mixture of ovalbumin, α-chymotrypsinogen, cytochrome c, ribonuclease A and lysozyme was used to assess the chromatographic performance for large molecules while a cytochrome c digest served as a model mixture of peptides. All tested columns featured excellent mass transfer as demonstrated with very steep breakthrough curves. The highest binding capacities were found for columns prepared using the two step functionalization. Columns with sulfonic acid functionalities adsorbed up to 21.5 mg/mL lysozyme while the capacity of the weak cation exchange column functionalized with acrylic acid was 29.2 mg/mL.