2020

M. Morani, T.Duc Mai, Z. Krupova, P. Defrenaix, E. Multia, M. Riekkola, M. Taverna

Analytica Chimica Acta 1128 (2020) 45-51

Abstract

This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles’ (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an ‘inorganic-species-free’ background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method method reached 8 × 10⁹ EVs/mL, whereas the calibration curve was acquired from 1.22 × 10¹⁰ to 1.20 × 10¹¹ EVs/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method.

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Pete Gagnon, Katja Vrabec, Tjaša Lojpur, and Aleš Štrancar

BioProcess International, 18 (4) April 2020

Exosomes are a subject of rapidly growing therapeutic interest in the biopharmaceutical industry for two principal reasons. The first reason is that they are the primary communicators of instructions from source cells to target cells. Exosome surface features define their destination. They recognize complementary features on target cells, dock with them, and deliver their programmed instructions in the form of microRNA. The second reason is that exosomes are immunologically silent. As normal human cell products, and by contrast with gene therapy vectors such as virus particles, exosomes bypass the issue of triggering an immune response that might interfere with therapy.

Source cells include stem cells, which is why exosomes are of particular interest in the field of regenerative medicine. Recent research documenting the ability of exosomes to reverse the effects of severe strokes highlights their potential. It also underlines the need for scalable purification technology to advance these products through clinical trials and on to licensed manufacture. A platform approach was a major factor in the initial and continuing success of monoclonal antibodies. Exosomes likewise represent an extended family of individual products with similar properties. It stands to reason that a platform approach will prove equally valuable for exosomes. In this article we describe initial efforts toward that goal.

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Hietala V, Horsma-Heikkinen J, Carron A, Skurnik M, Kiljunen S.

Frontiers in microbiology vol. 10 1674. 23 Jul. 2019

Abstract

The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiM_fHyAci03, and Staphylococcus phage vB_SauM_fRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_fHoEco02 lysate from 3.5 × 104 Endotoxin Units (EU)/109 plaque forming units (PFU) to 0.09 EU/109 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/109 PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/109 PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.

Keywords: antibiotic resistance, bacteriophage, phage therapy, endotoxin, enterotoxin

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2019

Calef Sánchez-Trasviña, Marco Rito-Palomares, and José González-Valdez

Advances in Polymer Technology, Volume 2019, December 12 2019, 10 pages

Abstract

PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.

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Wang Chunlei, Mulagapati Sri Hari Raju, Chen Zhongying, Du Jing, Zhao Xiaohui, Xi Guoling, Chen Liyan, Linke Thomas, Gao Cuihua, Schmelzer Albert, Liu Dengfeng

Molecular Therapy  Methods & Clinical Development, Volume 15, September 26 2019, Pages 257-263

Abstract

Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 1011 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.

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Keywords: AAV, AAV6.2, Chromatography, Anion exchange chromatography (AEC), Empty capsids, AUC, High-throughput

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Katarina Marković, Radmila Milačič, Janja Vidmar, Stefan Marković, Katja Uršič, Martina Nikšić Žakeljc, Maja Cemazar, Gregor Sersa, Mojca Unk, Janez Ščančar

Journal of Trace Elements in Medicine and Biology, Volume 57, January 2020, Pages 28-39.

Abstract

Monolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run. Here, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. The data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. Low-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.

Keywords: low-pressure and high-pressure conjoint liquid chromatography, anion-exchange and affinity monolithic disks, inductively coupled plasma mass spectrometry, Pt-based chemotherapeutics, serum of cancer patients

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Sofiya Fedosyuk, Thomas Merritt, Marco Polo Peralta-Alvarez, Susan J. Morris, Ada Lam, Nicolas Laroudie, Anilkumar Kangokar, Daniel Wright, George M. Warimwe, Phillip Angell-Manning, Adam J. Ritchie, Sarah C. Gilbert, Alex Xenopoulos, Anissa Boumlic, Alexander D. Douglas

Vaccine (2019).
Published online 30 April 2019.

A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating  adenovirus vectors, but important challenges remain. There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using  commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.
Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell – promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype.

Keywords: Simian adenovirus, GMP, Clinical trials, Single-use, Biomanufacturing, Bioreactor, Purification

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Evgen Multia, Crystal Jing Ying Tear, Mari Palviainen, Pia Siljander, Marja-Liisa Riekkola

Analytica Chimica Acta (2019).
Published online 2019 Sep 11.

A new, fast and selective immunoaffinity chromatographic method including a methacrylate-based convective interaction media (CIM®) disk monolithic column, immobilized with anti-human CD61 antibody, was developed for the isolation of CD61-containing platelet-derived extracellular vesicles (EVs) from plasma. The isolated EVs were detected and size characterized by asymmetrical flow field-flow fractionation (AsFlFFF) with multi-angle light-scattering (MALS) and dynamic light-scattering (DLS) detection, and further confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The isolation procedure took only 19 min and the time can be even further decreased by increasing the flow rate. The same immunoaffinity chromatographic procedure, following AsFlFFF allowed also the isolation and characterization of platelet-derived EVs from plasma in under 60 min. Since it is possible to regenerate the anti-CD61 disk for multiple uses, the methodology developed in this study provides a viable substitution and addition to the conventional EV isolation procedures.

Keywords: Immunoaffinity chromatography, Isolation, Monolithic disk column, Extracellular vesicles, Platelet-derived vesicles, CD61

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J. R. Lorsch, A. M. Munoz, J. S. Nanda, V. Rajagopal, P. Yourik, S. E. Walker

RNA Biology (2017), volume 14 (2), pp. 188–196.
Published online 2016 Dec 16.

In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

KEYWORDS: Eukaryotic translation, in vitro translation, ribosome, ribosome purification, yeast

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Thanaporn Liangsupree, Evgen Multia, Jari Metso, Matti Jauhiainen, Patrik Forssén, Torgny Fornstedt, Katariina Öörni, Aleš Podgornik & Marja-Liisa Riekkola 

Scientific Reports, volume 9, August 2019

Low-density lipoprotein (LDL) is considered the major risk factor for the development of atherosclerotic cardiovascular diseases (ASCVDs). A novel and rapid method for the isolation of LDL from human plasma was developed utilising affinity chromatography with monolithic stationary supports. The isolation method consisted of two polymeric monolithic disk columns, one immobilized with chondroitin-6-sulfate (C6S) and the other with apolipoprotein B-100 monoclonal antibody (anti-apoB-100 mAb). The first disk with C6S was targeted to remove chylomicrons, very-low-density lipoprotein (VLDL) particles, and their remnants including intermediate-density lipoprotein (IDL) particles, thus allowing the remaining major lipoprotein species, i.e. LDL, lipoprotein(a) (Lp(a)), and high-density lipoprotein (HDL) to flow to the anti-apoB-100 disk. The second disk captured LDL particles via the anti-apoB-100 mAb attached on the disk surface in a highly specific manner, permitting the selective LDL isolation. The success of LDL isolation was confirmed by different techniques including quartz crystal microbalance. In addition, the method developed gave comparable results with ultracentrifugation, conventionally used as a standard method. The reliable results achieved together with a short isolation time (less than 30 min) suggest the method to be suitable for clinically relevant LDL functional assays.

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Dr. Xiaotong Fu, Dr. Wei-Chiang  Chen, C. Argento, R. Dickerson, P. Clarner, V. Bhatt, G. Bou-Assaf, Dr. M. Bakhshayeshi, Dr. Xiaohui Lu, Dr. S. Bergelson, Dr. J. Pieracci

Human Gene Therapy (2019)

Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids—viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography (AEX-HPLC) assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability to both the transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC) methods used in empty-to-full capsid ratio quantification, yet providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes (vg)/ml.

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K. Trabelsi, M. Ben Zakour, H. Kallel

Vaccine (2019)

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease.
Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield.
 

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This discussion introduces new analytical approaches that enable in-line chromatographic detection of exosomes. One approach can discriminate extracellular vesicles from nonvesicle contaminants, and one potentially can discriminate exosomes from other vesicles. Examples illustrate how they enable development of more effective and better documented purification methods. The special qualifications of monolithic chromatography media for exosome purification are discussed. New process tools designed to accommodate some of the special challenges of exosome purification are introduced.

Feel free to download the eBook by clicking on the link to attachment below.

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2018

The purpose of this book is to provide you with a guide to developing monoclonal antibody purification procedures taht meet the requirements of both research and commercial applications. It is based on successful purifications developed for over 250 monoclonal-based products, addressing a wide range of diagnostic and therapeutic applications. it is supported by nearly 1000 citations from the scientific literature and enriched by the insights of skilled practitioners from throught the industry. It incorporates over 100 figures and tables to illustrate key concepts.

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Laura M. Fischer, Michael W. Wolff, Udo Reichl, Vaccine 2017 July 17

The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA. The continuous chromatography was implemented as simulated moving bed chromatography (SMB) in a three zone open loop configuration with a detached high salt zone for regeneration.
SMBs exploiting 10% and 50% of the monoliths’ dynamic binding capacity, respectively, allowed the depletion of >98% of the DNA and >52% of the total protein. Based on the hemagglutination assay (HA assay), the virus yield was higher at 10% capacity use (89% vs. 45%). Both SMB  separations resulted in a ratio of total protein to hemagglutinin antigen (based on single radial diffusion assay, SRID assay) below the required levels for manufacturing of human vaccines (less than 100 mg of protein per virus strain per dose). The level of contaminating DNA was five-times lower for the 10% loading, but still exceeded the required limit for human vaccines. A subsequent Benzonase treatment step, however, reduced the DNA contamination below 10 ng per dose. Coupled to continuous cultivations for virus propagation, the establishment of integrated processes for fully continuous production of vaccines seems to be feasible.

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Miladys Limonta, Lourdes Zumalacarregui, Urska Vidic, Nika Lendero Krajnc

The main component of the Center for Genetic Engineering and Biotechnology (CIGB) candidate vaccine against Hepatitis C virus (HCV) is the pIDKE2 plasmid. The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharamceutical-grade plasmid DNA (pDNA)with 95% purity. The advantages of this procedure include high plasmid purity and the elimination of undesirable additives. such as toxic organic extractants and animal-derived enzymes. However, yields and consequently the productivity of the process are low. Previous work demonstrated that the most critical step of the process is the reverse phase chromatography, where conventional porous particle resins are used. Therefore, to increase the process productivity alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step. Here, a comparison between the behaviours of CIM® C4-HLD and Sartobind phenyl matrices was performed.

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Tsutomu Arakawa, Pete Gagnon

Journal of Pharmaceutical Sciences 107 (2018) 2297-2305

The concept of cosolvent exclusion was developed by a group of Timasheff's laboratory in 1970-1990 and is currently used widely to explain the effects of a variety of cosolvents on the stability and solubility of macromolecules. Not surprisingly, these concepts have had substantial influence in the fields of formulation, protein folding and unfolding, but they have perhaps more surprisingly found their way into the field of chromatography. A variety of excluded cosolvents have been used to enhance binding and resolution of proteins and other macromolecules in ion exchange, hydroxyapatite, affinity, and hydrophobic interaction chromatography. These cosolvents include salting-out salts, amino acids and polymers, and frequently polyethylene glycol (PEG). A new mode of chromatography, termed “steric exclusion chromatography,” was recently introduced. It employs hydroxylated solid phase surfaces. Steric exclusion of the PEG stabilizes the association of macromolecules with the solid phase. Elution is achieved by reducing the PEG concentration. Magnetic particles are also used in this chromatography. This review summarizes the concepts of preferential cosolvent exclusion and its applications in column chromatography.

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2017

V.Rajamanickam, D.Wurm, C.Slouka, C.Herwig, O.Spadiut

Anal Bioanal Chem (2016)

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future

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Marina Naldi, Urh Černigoj, Ales Štrancar, Manuela Bartolini

Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5 mm×5.2 mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90 ± 9 s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200 ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.

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Alicia T Lucero, Sergio A Mercado, Anamaría C Sánchez,Carolina A Contador, Barbara A Andrews and Juan A Asenjo, Journal of chemical technology and biotechnology, (2017)

BACKGROUND: Gene therapy is a potent alternative for long-lasting inhibition of alcohol consumption. This study compares the purification of a recombinant adenoviral vector serotype 5 (rAdV5) for use in gene therapy against alcoholism using two anion-exchange methods.

RESULTS: Two anion-exchange chromatography methods using fast protein liquid chromatography were compared using a packed-bed column (Q-Sepharose™ XL) and two monolithic columns (CIM™ QA-1 and CIM™ DEAE-1). An improved and reproducible separation of recombinant adenovirus type 5 from cell lysate contaminants was achieved using the two strong anion-exchange columns in a two-step gradient chromatography. Higher adenovirus yields were achieved using the CIM QA-1 tube monolithic column at sample volumes of both 1 and 10 mL compared with the Q-Sepharose XL column. At higher flow rates, the CIM QA-1 tube monolithic column achieved better separation of the target fraction. Process recovery was improved from 28% using the Q-Sepharose XL column to 34% with the CIM QA-1 tube monolithic column quantified as vector genome. Analysis by SDS-PAGE demonstrated a purity of 70% for purified adenovirus using the CIM QA-1 tube monolithic column.

CONCLUSION: This study indicated that the use of a CIM QA-1 tube monolithic column is a better alternative than Q-Sepharose XL, and CIM DEAE-1 tube monolithic columns for the primary purification process of rAdV5 carrying the human aldehyde dehydrogenase-2 antisense gene. This purification strategy has been used as a basis to scale-up a GLP process for the production of material at the National Research Council of Canada to be used in preclinical trials of this gene therapy against alcoholism

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