Over the last years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce non-dividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the purification and concentration of high titer and high quality vector stocks is still time-consuming and scale-limited. We aimed to develop a simple and cost-effective capture purification step capable of separating the produced lentiviral vectors from the preparation originally containing a load of recombinant baculoviruses used to transiently transfect 293T producer cells. Even though recombinant baculoviruses do not present major safety concerns1, the final product (purified lentiviral vectors) should be pure enough to be tested in (pre-)clinical studies2. A capture step has been preliminarily evaluated. Both lentiviruses and baculoviruses are enveloped, thus per se prone to degradation through processing. Furthermore, both show overall surface negative charges at physiological pH3,4. As such, our rationale was to use an anion-exchange bind-elute step with enough resolution to separate the two viruses upon elution. It was likely that the difference in the overall electrostatic charges of the two viruses can be used to our advantage if a sufficiently extended salt elution gradient is used.
Biomanufacturing of antibodies, therapeutic proteins and vaccines or gene delivery vectors (either DNA or virus based) is a very complicated process where many things can go wrong. This is even more pronounced as the target biomolecules are extremely susceptible to the environmental conditions both during cultivation (upstream processing) as well as during isolation and purification (downstream processing). One can always doubt whether we have enough information about our complex biomolecule samples to consistently develop a safe product by running a robust and efficient purification bioprocess.
By using and understanding novel technologies one can design new process analytic technology (PAT) initiatives to overcome some of these problems. Here, we present novel monolithic analytical columns — CIMac columns — that can bridge this gap. In the first example, CIMac columns were applied for monitoring the purification process of virus like particles (VLP) which are used for production of vaccines and as delivery systems in gene therapy. In the second example, the monolithic analytical columns were also applied for monitoring the fermentation process of bacteriophages.
Avir Green Hills Biotechnology is developing innovative seasonal and pandemic influenza vaccines based on the deletion of the NS1 gene (del NS1 vaccine).The vaccine is replication-defective and applied intranasally. Recently,clinical phase I studies for H1N1 monovalent vaccine and H5N1 avian influenza vaccine were completed. Both were confirmed to be safe and immunogenic for humans. A production and purification process, which was successfully employed for the pilot-scale production of H1N1 and H5N1 influenza A vaccine virus, will be presented and compared to standard ultracentrifugation method. Details on obtained life virus yields as well as impurity removal will be given. The vaccine virus is produced in static cell culture using Vero (African Green monkey kidney) cells. After clarification the vaccine virus bulk is purified using the same(chromatography-based) scheme for all different subtypes: Concentration by tangential ultrafiltration, AEX chromatography using a CIM QA monolith, and an SEC polishing step allowing for buffer exchange. This purification scheme guarantees the thorough depletion of host cell DNA and total protein. For the ultracentrifugation approach chromatographic steps were replaced by a gradient ultracentrifugation step, comparison data are shown. In addition, an HPLC method for quantifying influenza virus in the vaccine with the use of CIM monolithic columns will be presented and the results will be compared with haemagglutination method.
Rabies virus cause acute encephalitis. It is widely distributed around the globe and more than 55,000 people perish yearly and an additional 10 million post-exposure treatment are reported. About 95% of human deaths occur in Asia and Africa. In countries that are endemic to rabies an immense need for cost-effective large-scale production of the Rabies vaccine occurs. Achieving required quality is challenging because majority of rabies vaccines are produced in Vero cells. This makes Rabies vaccine difficult to manufacture due to low titre of vaccine with lots of residual cellular DNA and serum proteins.
The objective of this work was to improve purity of rabies vaccine regarding residual DNA presence. Different mobile phases with different pH values were explored. Moreover, to develop cost-efficient downstream process for Rabies vaccine, monolith-based purification step was performed in different stages of downstream processing. Chromatographical fractions were analyzed for efficiency of DNA removal. In addition, recovery of Rabies vaccine was monitored. Finally, knowing the optimal conditions, a step-wise gradient was used for purification of larger amount of Rabies vaccine.
Adeno-associated virus (AAV) vectors continue to hold immense promise as gene transfer vehicles for a variety of gene therapy applications. Numerous pre-clinical and human clinical studies have been undertaken with rAAV, employing several of the identified serotypes to leverage their differing tissue tropism to correct a broad spectrum of genetic diseases. Despite the advantageous characteristics of rAAV and the extensive research into pre-clinical applications, production and purification scale-up continues to limit recombinant AAV (rAAV) use in large clinical trials that require even moderate vector doses. Therefore, AGTC has developed a high-yielding, scalable rAAV production system in suspension BHK cells that employs co-infection with two hybrid rHSV-rAAV vectors to provide all cis and trans-acting rAAV elements and the requisite helper virus functions for rAAV manufacturing.
In contrast to traditional, resin-based chromatography methods for rAAV purification, we have developed a two-step chromatographic process that employs a novel anion exchange Convective Interaction Media® monolithic column (CIM® monolith, BIA Separations) capture step followed by affinity chromatography (AVB Sepharose™, GE Healthcare), which yields rAAV vector stocks in very high purity. This scalable process allows significant reduction in processing time due to the high capture step dynamic binding capacity, flow rates and resolution. The resulting overall chromatography recovery compares favorably to our first and second generation processes which used three-step, resin-based column chromatography and membrane-based two step chromatography, respectively.
The CIM QA-AVB process was scaled to accommodate 10 L suspension production runs and was successful at recovering as much as 1 × 1015 purified AAV1 DRP in a single day. The process is highly reproducible and it is applicable for the purification of multiple AAV serotypes with over 95% purity and overall yield of > 30%.
Bacteriophages were in recent years identified as a useful potential tool for different biotechnological applications such as alternative to antibiotics, detection of pathogenic bacteria, delivery vehicles for protein and DNA vaccines and as gene therapy delivery vehicles (1). For all listed fields of use it is important that phages are highly purified with preserved biological activity. Phage and other virus purification have traditionally been carried out by CsCl density gradient ultracentrifugation, which is however difficult to be scaled-up. An alternative is chromatography already proved to be efficient for purification and concentration of certain virus types.
One of the key issues using chromatography is processing time and capacity of the resin. Novel type of chromatographic resin named monoliths was already proved to be very efficient for fast separation and purification of macromolecules as are large proteins, DNA and viruses (2,3,4).
Our aim was to investigate whether Convective Interaction Media (CIM) methacrylate monolithic columns can be implemented for purification and concentration of phage T4 (virus for E.coli). Chromatographic method using linear gradient was implemented to investigate conditions for phage elution and to establish the optimized chromatographic method applying step gradient. We analyzed phage recovery and purity together with method reproducibility.
Avir Green Hills Biotechnology is developing innovative seasonal and pandemic influenza vaccines based on the deletion of the NS1 gene (delNS1 vaccine). The vaccine is replication-defective and applied intranasally. Currently, an H1N1 monovalent vaccine is being tested in a clinical phase I study, with an H5N1 avian influenza vaccine soon to be initiated. A production and purification process, which was successfully employed for the pilot-scale production of H1N1 and H5N1 influenza A vaccine virus, will be presented. Data on the selection of chromatographic media, relevant to eliminate downstream purification bottlenecks will also be discussed.
Details on obtained virus yields as well as impurity removal will be given. The vaccine virus is produced in static cell culture using Vero (African Green monkey kidney) cells. After clarification the vaccine virus bulk is purified using the same scheme for all different subtypes: Concentration by tangential ultra filtration, AEX chromatography using a CIM QA monolith, and an SEC polishing step allowing for buffer exchange. This purification scheme guarantees the thorough depletion of host cell DNA and total protein. In addition, an HPLC method for quantifying influenza virus in the vaccine with the use of CIM monolithic columns will be presented and the results will be compared with haemagglutination method.
In an average influenza season, we face hundreds of thousands of influenza cases. Up to 50,000 deaths per year can be ascribed to influenza epidemics. Nevertheless, this is relatively harmless compared to the current, permanent threat of a worldwide pandemic caused by avian influenza.
AVIR Green Hills Biotechnology is developing innovative seasonal and pandemic influenza vaccines based on the deletion of the NS1 gene (ΔNS1 vaccine) . The vaccine is replication-defective and applied intranasally. Currently, an H1N1 monovalent vaccine is being tested in a clinical phase I study and clinical trials with H5N1 avian influenza vaccine will follow in fall 2007.
A production process, which was successfully employed for the pilot-scale production of H1N1 and H5N1 influenza A virus is presented here. The upstream process is performed according to the specific requirements of the respective influenza subtypes. Currently, 15 L batches are produced in cell factories using Vero (African green monkey kidney) cells. The vaccine bulk is purified by using the very same scheme for all different subtypes. For purification, the cell culture supernatant is clarified by centrifugation and the virus is concentrated by tangential ultra filtration. The concentrated virus is subsequently purified in two chromatographic steps which were co-developed with BIA Separations d.o.o.: First, an anion exchange monolithic column is used. This is followed by size exclusion chromatography for polishing and buffer exchange.
This purification scheme guarantees the thorough depletion of host cell DNA and total protein, and recovers at least 25% of the infectious virus.
During last decades different methods for purification of influenza viruses have been described. Most of these methods were developed for purification of egg derived influenza virus which is still the main production system for influenza vaccine viruses. Since cell culture based technology is gaining more and more importance, the need for alternative, efficient and scaleable purification methods has risen. Chromatography is becoming a method of choice for purification of viruses. Relevance of this technique was recently demonstrated also for influenza viruses. Methacrylate monoliths are characterized by large channel diameter, high surface accessibility and convective mass transport. As a consequence they have high binding capacity for large molecules, enable high flow rates at low pressure drop and therefore increase productivity. Recently it has been proven that methacrylate monolithic columns can also be used for purification and concentration of different viruses.
It was the purpose of this work to explore possibilities for purification of influenza viruses on ion exchange methacrylate monoliths. Different subtypes of influenza A and influenza B virus were tested employing various ion exhange monolithic columns.
During the last decade important developments in molecular medicine and adenoviral vector design have been achieved, leading to an increased use of adenoviral vectors in clinical gene therapy protocols. One of the main advantages of the adenovirus is their ability to replicate at high titres in permisive cell lines. The availability of large quantities of adenoviral vector preparations is recognized as an important limitation to pre-clinical and clinical studies. Consequently there is a global focus on large scale production of adenoviral vectors, providing high titres combined with fast, effective and reliable purification methods.
Viruses have proven to be useful vectors for gene therapy purposes. As therapeutics for human use they must be pure and contaminant free. Traditionally, viruses are purified by complicated and time consuming methods such as CsCl density gradient centrifugation or similar. In recent years liquid chromatography has became interesting method for virus purification. It provides high level of purity required for human use and increases productivity. Traditional chromatographic supports were mostly designed for purification of proteins and as such are commonly inappropriate for viruses. Alternative to traditional chromatographic support are methacrylate monoliths (CIM monoliths), characterized by large channel diameter, high surface accessibility and convective mass transport.
The aim of this work was to characterize CIM supports for separation and possible purification of a model virus Tomato mosaic virus (ToMV) from crude plant material.
Traditionally, viruses are purified by time consuming methods such as CsCl density gradient centrifugation or similar. These methods are often inefficient and limited to small scale. In recent years different methods for virus purification, based on ion exchange, gel filtration and affinity chromatography have became popular. Recently, CIM® disk monolithic columns were used for successful concentration of two plant viruses (1) and for improved detection of two human viruses (2). Cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV) were concentrated and subsequently detected from extremely diluted samples in which they were initially undetectable. Successful concentrations of both viruses encourage us to explore the possibilities of CIM® supports for virus purification. As a model virus ToMV was selected. ToMV is a rod shaped plant virus with a typical size of 300 x 18 nm and isoelectric point at pH 4.6.
Gene therapy which is becoming more and more important in human health care requires the purification of high molecular mass compounds, so called nanoparticles (e. g. viruses and plasmids). The method of choice to ensure proper purity would be chromatography.
Most of the chromatographic supports available on the market at the moment can not follow the requests for such work due to low binding capacity for large molecules, limitation with regards to the time of the separation process and requests for CIP (cleaning in place) and SIP (sanitation in place).
Monolithic supports represent a new generation of chromatographic supports. In contrast to conventional particle supports, where the void volume between individual porous particles is unavoidable, these supports consist of a single monolith highly interconnected with larger and smaller open flow-through channels. Due to the structure, molecules to be separated are transported to the active sites on the stationary phase by convection, resulting in very short separation times. This is especially true for large molecules.
In this work we will present the use of monolithic supports for the separation of different nanoparticles on analytical and preparative scales. It will be shown that monolithic supports can overcome the limitations of particle-based supports for the analytics and isolation of big molecules and represent a major step towards the safe and efficient purification or production of nanoparticles.
Traces of DNA in RNA samples represent impurities that could affect results of mRNA quantification and cDNA synthesis. In most cases, the DNA impurities in RNA samples are removed using enzyme deoxyribonuclease (DNase), which specifically breaks down DNA. In order to avoid the addition of DNase into the analyzing sample, the use of immobilized DNase on solid support is recommended. Because of the DNA size, very few supports available on the market enable efficient interaction between immobilized enzyme and DNA.
In recent years a new group of supports named monoliths was introduced. Because of enhanced exchange between mobile and stationary phase separation and bioconversion processes are significantly accelerated. Therefore also the efficiency of DNA removal using immobilised enzyme might be competitive to the degradation with free enzyme.
Strains of the anaerobic bacterial genus are thought to play an important role in fiber degradation. sp. Mz5 was previously isolated from the rumen of a black and white Friesian cow and its xylanolytic activity was proved to be at least 1,65 times higher than the activities of all of the compared well known xylan-degrading rumen bacterial species and strains (1). High xylanolytic activity was the reason for partial isolation of its xylanases in order to study their special characteristics and possible biotechnological applications later.
High performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic separation of plasmid DNAconformations. However, no study about the separation of small molecules was performed until now. In this work, we investigated the possibility of gradient and isocratic separations of small molecules with Convective Interaction Media (CIM) disks of different chemistries. We proved that it was possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed phase mode. Fairly good separation of four oligonucleotides could be achieved on the disk of 0.3 mm thickness. The effect of the gradient parameters on the resolution in the case of gradient mode was studied and compared with the separation under isocratic conditions.
It was shown that similar peak resolution can be achieved in both gradient and isocratic modes. In addition, it was found that the flow rate does not have a pronounced influence on the resolution in the flow rate range between 1 and 10 mL/min. However, it seems that the resolution with the flow rate even slightly increases as a consequence of the increased pore accessibility. In accordance with conventional particle HPLC columns, the resolution increases with the monolith thickness. On the other hand, the mobile phase composition has to be carefully adjusted to obtain optimal resolution, especially in the case of isocratic separations. Because of this feature, CIM monoliths seem to be competitive to other, commercially available stationary phases.
Organic acids are important metabolites of several biochemical pathways in microorganisms and as such they are frequent main or by-products in different bioprocesses. Consequently, a demand for their monitoring is often present. One of the most applied methods for organic acids determination is certainly HPLC using different separation mechanisms such as reversed-phase, ion-exchange or ion-exclusion chromatography, all based on separation under isocratic flow conditions. To achieve the isocratic separation, multiple steps of adsorption-desorption process are needed and therefore conventional chromatographic columns with long layer of separation material were considered as a necessary tool for achieving this effect.
Recently, it was shown that isocratic separation could also be performed on thin monolithic layers. The isocratic separations of plasmid DNA conformers (1), oligonucleotides (2, 3) and peptides (3) in the ion-exchange mode were demonstrated as well as isocratic reversed-phase separation of a mixture of steroids was obtained (3) all on thin GMA-EDMA monoliths commercially available under trademark CIM™ (Convective Interaction Media). The results indicated the possibility of applying CIM™ monolithic columns also for isocratic separation of some other small charged molecules. Since the average analysis time using CIM™ disk monolithic columns is up to a few minutes, these supports can be a material of choice for separation of organic acids.
CIM® supports are novel monolithic chromatographic supports. In contrast to conventional particle based chromatographic supports they consist of a single porous polymer. The pores form a highly interconnected network, which enables the flow of the mobile phase through the monolith. Molecules to be separated are transported to the surface by the convection. Since the diffusion is not a bottleneck any more, also the resolution and the dynamic capacity of the monolith are flow independent and an average analysis time is typically below one minute. Furthermore, CIM® columns were successfully applied for the purification of proteins directly from the fermentation broth.
Manganese peroxidases (MnP) and lignin peroxidases (LiP) are a family of glicosilated hemo-proteins, which are excreted into the growth medium during the idiophasic growth of the white rot fungus Phanerochaete chrysosporium. They are both involved in the lignin degradation. For their analysis and separation from the growth medium, HPLC is commonly applied. Besides the separation by Na-acetate concentration gradient (2), also the chromatofocusing can be used (3). A fast method for LiP isoenzyme separation from the growth medium of P. chrysosporium using CIM™ QA disk monolithic columns has been recently developed (1). A modified method was tested on the growth medium containing MnP isoenzymes.
The aim of our work was to study the direct monitoring and purification of proteins from the fermentation broth using ion-exchange CIM® supports. Therefore, we studied the possibility of monitoring and purifying lignin peroxidase extracelular protein isoforms produced by the fungus Phanerochaete chrysosporium. These isoenzymes which also differ in their catalytic properties are able to partially depolymerize lignin and to oxidise several xenobiotics.
The white rot fungus Phanerochaete chrysosporium under nitrogen or carbon limitation produces extracellular lignin peroxidases (LiP). They are able to partially depolymerize lignin and to oxidize several xenobiotics (DDT, PCB, PAH, etc.). By HPLC separation and isoelectric focusing multiple molecular forms of LiP have been isolated from the culture filtrate. For the isolation of LiP from the growth medium, mostly the HPLC technique with ion exchange Mono-Q or DEAE columns is used. The medium should be dialyzed before separation and usually also concentrated. Medium freezing is used to remove mucilaginous polysaccharides which disturb separation. The whole procedure is time consuming and information about isoenzyme content and their relative amounts in the growth medium is delayed for at least 1 day. HPLC separation itself lasts nearly an hour. For the separation of LiP isoenzymes from the culture filtrate, we used the monolithic stationary phase with weak (DEAE-diethylamine) and strong (QA-quaternary amine) ion exchange groups commercially available under trademark CIM (Convective Interaction Media). CIM supports are glycidyl methacrylate based monolithic porous polymer supports. As such they differ from conventional particle shaped chromatographic supports. The liquid is forced to flow through the support channels. Molecules to be separated are transported mainly by convection resulting in travelling times shorter for at least an order of magnitude. As a consequence the resolution as well as the binding capacity remain unaffected with the flow rate and a shorter analysis time can be achieved. This effect is even more pronounced in the case of large molecules such as proteins, which have a low diffusion coefficient. As such, CIM units can be advantageous also for lignin peroxidase isoenzymes separation and purification.