Publications
2010
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.
H. P. Lesch, A. Laitinen, C. Peixoto, T. Vicente, K.-E. Makkonen, L. Laitinen, J. T. Pikkarainen
Gene Therapy advance online publication, 20 January 2011
Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.
I. Gutierrez-Aguirrea, A. Steyer, M. Banjac, P. Kramberger, M. Poljšak-Prijatelj, M. Ravnikar
Journal of Chromatography A, 1218 (2011) 2368-2373
Rotaviruses are the leading cause of gastroenteritis in children and they exist widely in water environments. Ingestion of 10–100 viral particles is enough to initiate disease, what calls for extremely sensitive detection methods. In this study we have confirmed the validity of a recently published method for rotavirus concentration and detection based on the combination of methacrylate monoliths and real-time reverse transcription-quantitative PCR (RT-qPCR). The method was used to concentrate rotaviruses from different tap water and environmental water samples collected in Slovenia within years 2007 and 2009. The performance of virus concentration using monolithic supports was improved in comparison to the one of tangential ultrafiltration upon application of both methods on a range of environmental samples. Several samples were successfully concentrated on-site after successful adaptation of the method to field requirements. In such on-site format, the combination of concentration using CIM and detection using RT-qPCR detected as low as 30 rotavirus particles/ml, spiked in an environmental water sample.
L. Urbas, B. Košir, M. Peterka, B. Pihlar, A. Štrancar, M. Barut
Journal of Chromatography A, 1218 (2011) 2432-2437
Monoliths are chromatographic stationary phases, which were specially designed for efficient purification of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60 × 1010 viral particles/mL to at least 1.64 × 1011 viral particles/mL, the limit of detection was found to be 2.56 × 109 virus particles/mL and the limit of quantification was 5.12 × 109 virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus.
F. Smrekar, M. Ciringer, A. Štrancar, A. Podgornik
Journal of Chromatography A, 1218 (2011) 2438-2444
Binding of three different bacteriophages (phages), namely T7, lambda and M13 on methacrylate monoliths was investigated. Phage M13 exhibited the highest dynamic binding capacity of 4.5 × 1013 pfu/mL while T7 and lambda showed capacity of 1 × 1013 pfu/mL, all corresponding to values of around 1 mg/mL. Interestingly, capacity for lambda phage was increased 5-fold by increasing NaCl concentration in a loaded sample from 0 to 0.2 M while there was a constant capacity decrease for T7 and M13 phages. Under optimal conditions, recovery for all three phages approached 100%. Measurement of a pressure drop increase during loading enabled estimation of adsorbed phage layer thickness. At a maximal capacity it was calculated to be around 50 nm for T7 phage and 60 nm for lambda phage matching closely capside size thus indicating monolayer adsorption while 80 nm layer thickness was estimated for M13 phage showing its orientation along the pore.
P. Kramberger, R. C. Honour, R. E. Herman, F. Smrekar, M. Peterka
Journal of Virological Methods 166 (2010) 60–64166 (2010) 60–64
Bacteriophages (phages) are known to be useful in many fields from medicine to agriculture, and for a broad range of applications, including phage therapy and phage display. For some applications, especially in medicine, high purity and viability of phages are required. Methacrylate monoliths (Convective Interaction Media [CIM] monolithic columns), designed for purification of bionanoparticles, were applied for the purification of Staphylococcus aureus phages VDX-10 from bacterial lysate. With a single step purification method, more than 99% of host cell DNA and more than 90% of proteins were removed, with 60% recovery of viable phages. Comparable results were obtained when the purification method was scaled-up from a CIM monolithic disk to a larger CIM monolithic column. Additionally, the dynamic binding capacity of a methacrylate monolith column for S. aureus phages VDX-10 was determined.
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.
E.A. Ponomareva, V.E. Kartuzova, E.G. Vlakh, T.B. Tennikova
Journal of Chromatography B, 878 (2010) 567–574
The effect of different modes of α-chymotrypsin attachment to the surface of methacrylate-based ultrashort monolithic minicolumns on enzyme activity has been studied. The immobilization of protease was carried out via direct covalent binding of chymotrypsin, as well as via its attachment through small and polymer spacers. It was established that the lowest enzyme activity against N-benzoyl-l-tyrosine ethyl ester was found for bioreactor obtained via direct attachment of chymotrypsin to the surface of GMA–EDMA minidisks, whereas the highest parameter close to that determined for dissolved enzyme was found in the case of bioreactor prepared by the introduction of copolymer of 2-deoxy-N-methacryloylamido-d-glucose with N-vinylpyrrolidone and acrolein as a long and flexible polymer spacer. Additionally, the effect of flow rate of substrate recirculation on bioconversion efficiency was examined. Independently on immobilization method, the increase of flow rate led to the raise of biocatalytic efficiency.
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.
P. Gagnon
Roadmap to Process Development, issue 3/2010, Sartorius BIA Separations
Introduction
The first two articles in this series addressed column selectivity and capacity. This article discusses how to apply results from these preliminary studies to create fully functional multi-step purification procedures. The principles described here can be applied to proteins, plasmids, or virus particles.
Process modeling represents a nexus at which the theoretical ideals of purification meet the practical limitations of the laboratory, or in less elegant terms: where the rubber meets the road. The key theoretical principle is the notion of developing an orthogonal purification process. Orthogonal means pertaining to right angles. In purification terms, it translates to combining purification methods that are highly complementary to one another. Its value resides in the presumption that different purification methods bind the product by different sites, along with a unique subset of contaminants. The more complementary the methods, the lower the overlap in contaminant subsets, and the higher the purification factor offered by the particular combination of methods.
Attachments
2009
P. Gagnon
Roadmap to Process Development, issue 2/2009, Sartorius BIA Separations
Introduction
Determination of column loading capacity is a critical component of purification process development. Its most obvious link is to process economics, since the more product that can be loaded per unit of media volume, the smaller the column and volume of buffers, and the smaller the process footprint (manufacturing space requirement). But binding capacity is also linked directly to loading conditions, and beyond that, loading is a key determinant of purification performance and reproducibility. In practice, determination of optimal loading is tedious, time consuming, and expensive, especially due to the large amounts of sample it requires. This makes it all the more important to get it right the first time.
The objectives of this article are to highlight the process considerations that pertain to loading, and to provide you with a set of practical tools to determine capacity values that are meaningful in your particular usage context.
Attachments
P. Gagnon
Roadmap to Process Development, issue 1/2009, BIA Separations
Introduction
Commercial purification process development involves harmonizing a complex hierarchy of safety, regulatory, and economic considerations with the unique physicochemical characteristics of the product and the suite of contaminants that must be removed. This can be challenging even with product classes that exhibit fairly consistent chromatographic behavior, such as IgG monoclonal antibodies. It is even more demanding with products that do not support a platform approach. In either case, process development requires detailed knowledge of how the product behaves relative to contaminants within the operating ranges of the methods that may be used in its purification. This knowledge can be obtained only by characterizing product retention experimentally, a process that begins with initial screening. Screening produces the first indications of what methods offer the most promising fractionation capabilities, under what conditions, and in what order different methods may be linked together to yield an integrated multi-step purification procedure.
Attachments
E.G. Vlakh, T.B. Tennikova
Journal of Chromatography A, 1216 (2009) 2637-2650
Monolithic columns were introduced in the early 1990s and have become increasingly popular as efficient stationary phases for most of the important chromatographic separation modes. Monoliths are functionally distinct from porous particle-based media in their reliance on convective mass transport. This makes resolution and capacity independent of flow rate. Monoliths also lack a void volume. This eliminates eddy dispersion and permits high-resolution separations with extremely short flow paths. The analytical value of these features is the subject of recent reviews. Nowadays, among other types of rigid macroporous monoliths, the polymethacrylate-based materials are the largest and most examined class of these sorbents. In this review, the applications of polymethacrylate-based monolithic columns are summarized for the separation, purification and analysis of low and high molecular mass compounds in the different HPLC formats, including micro- and large-scale HPLC modes.
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.
L. Urbas, P. Brne, B. Gabor, M. Barut, M. Strlič, T. Čerk Petrič, A. Štrancar
Joural of Chromatography A, 1216 (2009) 2689-2694
Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A Sartorius BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.