Publications
2010
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.
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.
J. L. Ammerman, J. H. Aldstadt III
Microchim Acta (2009) 164:185-196
We describe the development and optimization of a sensitive and selective screening method for the measurement of trace levels of microcystins in surface waters. Several sample preparation techniques were compared, including solid-phase microextraction (SPME), particle-based solid-phase extraction (SPE), and monolith-based SPE. A flow-injection (FI) based approach employing a reversed-phase monolithic SPE column was found to be optimal. Quantification was performed by directly interfacing the FI-based SPE system to an electrospray ionization-mass spectrometer (ESI-MS). To more safely simulate peptidyl toxins such as the microcystins, a model peptide (i.e., angiotensin II) was used for method optimization. Sample loading flow rate and volume, eluent composition, and elution flow rate were optimized. Sample throughput was six samples per hour, a detection limit of 1.31 ng angiotensin II was demonstrated for a linear dynamic range from 1–1,000 ng and 3.4% relative standard deviation (n = 4, 100 ng sample). Sample volumes up to 1,000 ml of surface water could be loaded onto the monolithic SPE disk without exceeding the sorbent’s capacity. Unlike conventional particle-based SPE methods, the monolithic SPE disk does not need to be replaced between samples and could be used indefinitely. The FI-based SPE-ESI-MS method was successfully applied to the determination of microcystin-LR, the most common of the microcystins, in environmental samples and was demonstrated for the direct monitoring of chlorinated drinking water, with trends tracked over a period of eight months.
S. Yamamoto, M. Nakamura, C. Tarmann, A. Jungbauer
Journal of Chromatography A 1216 (2009) 2616-2620
Our previous study has shown that there is a good correlation between the number of charges of DNA (from trimer to 50-mer) and the number of binding sites B in electrostatic interaction chromatography (ion-exchange chromatography, IEC). It was also found that high salt (NaCl) concentration is needed to elute large DNAs (>0.6 M). In this paper we further performed experiments with large DNAs (up to 95-mer polyT and polyA) and charged liposome particles of different sizes (ca. 30, 50 and 100 nm) with a monolithic anion-exchange disk in order to understand the binding and elution mechanism of very large charged biomolecules or particles. The peak salt (NaCl) concentration increased with increasing DNA length. However, above 50-mer DNAs the value did not increase significantly with DNA length (ca. 0.65–0.70 M). For liposome particles of different sizes the peak salt concentration (ca. 0.62 M) was similar and slightly lower than that for large DNAs (ca. 0.65–0.70 M). The binding site values (ca. 25–30) are smaller than those for large DNAs. When arginine was used as a mobile phase modulator, the elution position of polyA and polyT became very close whereas in NaCl gradient elution polyT appeared after polyA eluted. This was mainly due to suppression of hydrophobic interaction by arginine.
2008
P. Gagnon
MSS2008
When monoclonal antibodies were first beginning to be commercialized, expression levels over 100 mg/L were considered outstanding, and cell culture was viewed as the bottleneck in manufacturing productivity. Antibody expression levels now commonly exceed 1 g/L and reports of 10 and 15 g/L have been recently announced. Downstream processing is now considered the bottleneck.
In one sense, the bottleneck is artificial. Cell culture production takes about two weeks (not counting preparation of seed stock) and purification takes about a week. In another sense, the bottleneck is real, and a genuine concern. Process time for the protein A capture step from 20,000 L of cell culture supernatant (CCS) commonly requires 72-96 hours. This represents multiple cycles. The long hold time for IgG produced in the early cycles increases the risk of degradation by proteolysis, deamidation, etc. It also increases the risk of contamination.
V. Frankovič, A. Podgornik, N. Lendero Krajnc, F. Smrekar, P. Krajnc, A. Štrancar
Journal of Chromatography A, 1207 (2008) 84–93(2008) 84 – 93
A weak ion-exchange grafted methacrylate monolith was prepared by grafting a methacrylate monolith with glycidyl methacrylate and subsequently modifying the epoxy groups with diethylamine. The thickness of the grafted layer was determined by measuring permeability and found to be approximately 90 nm. The effects of different buffer solutions on the pressure drop were examined and indicated the influence of pH on the permeability of the grafted monolith. Protein separation and binding capacity (BC) were found to be flow-unaffected up to a linear velocity of 280 cm/h. A comparison of the BC for the non-grafted and grafted monolith was performed using β-lactoglobulin, bovine serum albumin (BSA), thyroglobulin, and plasmid DNA (pDNA). It was found that the grafted monolith exhibited 2- to 3.5-fold higher capacities (as compared to non-grafted monoliths) in all cases reaching values of 105, 80, 71, and 17 mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1 M NaCl in the loading buffer. Recovery was comparable and no degradation of the supercoiled pDNA form was detected. Protein z-factors were equal for the non-grafted and grafted monolith indicating that the same number of binding sites are available although elution from the grafted monolith occurred at higher ionic strengths. The grafted monolith exhibited lower efficiency than the non-grafted ones. However, the baseline separation of pDNA from RNA and other impurities was achieved from a real sample.
A. Jungbauer, R. Hahn
Journal of Chromatography A, 1184 (2008) 62–79(2008) 62 – 79
Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8 L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses. These applications leverage monoliths’ inherent properties, such as fast operation and high capacity for large biomolecules. The height equivalent to a theoretical plate (HETP) and dynamic binding capacity do not change with velocity. This is explained by the convective transport through the channels with a diameter of above 1000 nm and has been experimentally verified and also supported by theoretical analyses. Despite low absolute surface area, these large channels provide enough area for adsorption of these large biomolecules, which cannot penetrate into conventional chromatography media designed for protein separation. Monoliths for preparative separations are mainly cast as polymethacrylate or polyacrylamide blocks and have been functionalized as ion exchangers or hydrophobic interaction chromatography media. So-called cryogels have channels more than 30 μm wide, enabling efficient processing of suspensions or even cell-chromatography. This review discusses the pressure drop characteristics, mass transfer properties, scale-up, and applications of monoliths in the context of conventional chromatography media.
E. S. Sinitsyna, E. N. Vlasova, E. G. Vlakh, T. B. Tennikova
Russian Journal of Applied Chemistry, 2008, Vol. 81, No. 8, pp. 1403–1409
Copolymers containing aldehyde, succinimidyl carbonate, and imidazolecarbamate groups were prepared by polymer-analogous transformations of epoxy groups of a macroporous monolithic polymeric support derived from glycidyl methacrylate and ethylene glycol dimethacyrlate. The effect of certain parameters on the course of the copolymer modification and immobilization of a protein on the surface of the polymeric support was studied. The possibility of using the matrices obtained for development of biorecognizing systems was examined.
M. Barut, A. Podgornik, L. Urbas, B. Gabor, P. Brne, J. Vidič, S. Plevčak, A. Štrancar
J. Sep. Sci. 2008, 31, 1867 – 1880
This review describes the novel chromatography stationary phase – a porous monolithic methacrylate-based polymer – in terms of the design of the columns and some of the features that make these columns attractive for the purification of large biomolecules. We first start with a brief summary of the characteristics of these large molecules (more precisely large proteins like immunoglobulins G and M, plasmid deoxyribonucleic acid (DNA), and viral particles), and a list of some of the problems that were encountered during the development of efficient purification processes. We then briefly describe the structure of the methacrylate-based monolith and emphasize the features which make them more than suitable for dealing with large entities. The highly efficient structure on a small scale can be transferred to a large scale without the need of making column modifications, and the various approaches of how this is accomplished are briefly presented in this paper. This is followed by presenting some of the examples from the bioprocess development schemes, where the implementation of the methacrylate-based monolithic columns has resulted in a very efficient and productive process. Following this, we move back to the analytical scale and demonstrate the efficiency of the monolithic column – where the mass transfer between the stationary and mobile phase is greatly enhanced – for the in-process and final control of the new therapeutics. The combination of an efficient structure and the appropriate hardware results in separations of proteins with residence time less than 0.1 s.
M. Ćurković Perica, I. Šola, L. Urbas, F. Smrekar, M. Krajačić
Journal of Chromatography A 1216 (2009) 2712-2716
A procedure based on Sartorius BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25 mM MOPS, 1.5 M NaCl, 0.1 mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis.
J. Ivancic-Jelecki, M. Brgles, M. Šantak, D. Forčić
Journal of Chromatography A 1216 (2009) 2717-2724
Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a Sartorius BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.
2007
R. Hahn, A. Tscheliessnig, P. Bauerhansl, A. Jungbauer
J. Biochem. Biophys. Methods 70 (2007) 87–94
Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.
R. Skudas, B. A. Grimes, E. Machtejevas, V. Kudirkaite, O. Kornysova, T. P. Hennessy, D. Lubda, K. K. Unger
Journal of Chromatography A, 1144 (2007) 72-84(2007) 72 - 84
In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of ∼25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18 < C8 < C4. The resolution of proteins and peptides by reversed-phase gradient liquid chromatography on n-octadecyl, n-octyl, and n-butyl bonded monolithic silica columns is optimized. The results obtained imply the use of acetonitrile concentration gradient up to 75% for n-octadecyl and n-octyl bonded monolithic silica columns, and the use of acetonitrile concentration gradient up to 85% for n-butyl bonded monolithic silica columns. With the respect to the gradient times and flow rates, the optimum conditions are the best with n-octyl and n-butyl bonded monolithic silica columns, where the range of optimum gradient times is up to ∼30 min and mobile phase flow rates in the range of 0.5–1 ml/min. Consequently, the best performance towards peak resolution is obtained with n-octyl bonded monolithic silica column with the respect to low concentration of organic phase gradient, fast separations and low solvent consumptions due to low flow rates.
J. Vidič, A. Podgornik, J. Jančar, V. Frankovič, B. Košir, N. Lendero, K. Čuček, M. Krajnc, A. Štrancar
Journal of Chromatography A, 1144 (2007) 63–71(2007) 63 – 71
Chemical and chromatographic stability of methacrylate-based monolithic columns bearing 3-N,N-diethylamino-2-hydroxypropyl (DEAE) and quarternary amine (QA) groups was studied. The leakage products from both monolithic columns were determined and the leakage of amines has been quantified in alkali solutions. Monolithic columns bearing QA functional groups being exposed to 1 M sodium hydroxide solution for up to 3 months caused reduction of ion-exchange groups for approximately 12%, while for DEAE monolithic columns was only around 3% in 1 year. In 0.1 M NaOH and 20% ethanol degradation was significantly lower. The main leaking compound from DEAE monolith was found to be 3-(diethylamino)-1,2-propanediol and 2,3-dihydroxypropyltrimethylammonium salt for QA monolith. During repeated 50 cleaning-in-place (CIP) cycles, no changes in chromatographic properties were detected.
B. A. Grimes, R. Skudas, K. K. Unger, D. Lubda
Journal of Chromatography A, 1144 (2007) 14-29(2007) 14-29
In this work, a parallel pore model (PPM) and a pore network model (PNM) are developed to provide a state-of-art method for the calculation of several characteristic pore structural parameters from inverse size-exclusion chromatography (ISEC) experiments. The proposed PPM and PNM could be applicable to both monoliths and columns packed with porous particles. The PPM and PNM proposed in this work are able to predict the existence of the second inflection point in the experimental exclusion curve that has been observed for monolithic materials by accounting for volume partitioning of the polymer standards in the macropores of the column. The appearance and prominence of the second inflection point in the exclusion curve is determined to depend strongly on the void fraction of the macropores (flow-through pores), (b) the nominal diameter of the macropores, and (c) the radius of gyration of the largest polymer standard employed in the determination of the experimental ISEC exclusion curve. The conditions that dictate the appearance and prominence of the second inflection point in the exclusion curve are presented. The proposed models are applied to experimentally measured ISEC exclusion curves of six silica monoliths having different macropore and mesopore diameters. The PPM and PNM proposed in this work are able to determine the void fractions of the macropores and silica skeleton, the pore connectivity of the mesopores, as well as the pore number distribution (PND) and pore volume distribution (PVD) of the mesopores. The results indicate that the mesoporous structure of all materials studied is well connected as evidenced by the similarities between the PVDs calculated with the PPM and the PNM, and by the high pore connectivity values obtained from the PNM. Due to the fact that the proposed models can predict the existence of the second inflection point in the exclusion curves, the proposed models could be more applicable than other models for ISEC characterization of chromatographic columns with small diameter macropores (interstitial pores) and/or large macropore (interstitial pore) void fractions. It should be noted that the PNM can always be applied without the use of the PPM, since the PPM is an idealization that considers an infinitely connected porous medium and for materials having a low (<6) pore connectivity the PPM would force the PVD to a lower average diameter and larger distribution width as opposed to properly accounting for the network effects present in the real porous medium.