Publications
2011
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.
F. Smrekar, M. Ciringer, J. Jančar, P. Raspor, A. Štrancar, A. Podgornik
Journal of Separation Science 2011, 34, 2152-2158
A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on-line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method.
C. Burden, J. Jin, A. Podgornik, D. G. Bracewell
Journal of Chromatography B, 880 (2012) 82- 89
Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25 mg/mL column.
L. Urbas, B. Lah Jarc, M. Barut, M. Zochowska, J. Chroboczek
Journal of Chromatography A, 1218 (2011) 2451-2459
Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.
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.
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.
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.
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.