In recent decades much work has been done on the development and optimisation of chromatographic supports in order to achieve efficient purification of biomolecules.
In the presented study we have investigated hydrodynamic and chromatographic properties of weak anion-exchange grafted monoliths (DEAE). Varying the concentration of the grafted polymer, grafted monoliths with different layer thickness and degree of branching were obtained. This results in a different hydrodynamic and chromatographic behavior of the examined monoliths such as permeability, ionic capacity and dynamic binding capacity (DBC) for the BSA protein. The DBC increases with the grafted layer thickness probably due to higher number of binding sites available for binding of the macromolecules. However, longer chains contribute to the reduction of the pore volume which results in a higher pressure drop. The latter can be additionally increased when biomolecules of interest are bound to the matrix. From this data information about the penetration depth into the grafted layer can be obtained giving an insight into the binding mechanism. Since the flow-unaffected properties were preserved even for large biomolecules, grafted monoliths may become a resin of choice for downstream processing of various macromolecules.
CIMac™ Analytical Columns are high-performance monolithic columns offering all the advantages of a special continuous short polymeric bed and are primarily intended for fast, efficient and reproducible separations of biomolecules like large proteins – antibodies (IgG, IgM), plasmid DNA, phages and viral particles. Their small volume and short column length allow the operation at high volumetric flow rates (from 1 to 30 column volumes/min) thus enabling receiving the information about the product quantity and purity in just a few minutes. These columns are pre-packed in dedicated stainless steel housings and allow user friendly connections to HPLC equipment. The product family offers strong cation exchange, strong and weak anion exchange and specialty analytical column for plasmid DNA. All columns can be effectively used for the in-process and final control of various samples from different purification process steps.
The biotechnological production of recombinants proteins consists of two main processes, upstream (biosynthesis) and downstream (protein purification) process. During the last decades the upstream process for mammalian cell culture has been improved significantly yielding in high amounts of protein. This development however led to a new challenge : the downstream process became a bottle-neck because of the large amounts of protein per batch in combination with the protein specific behaviors at high concentration.
In protein purification preparative chromatography is synonymous to “column chromatography”, and the favorable statics of a column are out of question for the physical requirements of beaded matrices. However, when approaching larger scales the physical dimensions of chromatography columns turn unfavorable: shallow gel beds of wide diameters. The footprint of such device increases drastically as does the weight, consequently resulting in limitations regarding floor space and floor bearing force.
A suitable chromatographic base matrix that is not obliged to a distinctive column design is a single piece of polymer – a monolith. Leaving the conventional column design, we have constructed a device for a monolith of rectangular shape, with the size of the monolith only limited by total weight (e.g. for handling and / or transportation). Using this design in a modular way, the individual modules can be stacked to make use of the height of a room at a very low footprint. A specific distribution system for feeding the monolith modules has been designed to allow a true linear scale-up from laboratory to large technical scale.
Application of plasmid DNA for gene therapy and vaccination has gained substantial interest in the last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Downstream processing has major influence on achieving regulatory demands in pDNA production and in order to get optimal purity different purification techniques have to be applied. It was demonstrated that methacrylate monoliths can be used for efficient purification process of plasmid DNA. High dynamic binding capacities and high flow rates of methacrylate monolith enable excellent purity and productivity.
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.
Anion-exchange chromatography is fundamental in downstream processing of plasmids both as a process and analytical technique. CIM anion-exchange monolithic columns have already been successfully used for the industrial scale purification of pharmaceutical grade small plasmid DNA .
In this work we report about the use of the newly developed monolithic analytical column intended for plasmid DNA determination in terms of its analytical performance. Higher degree of sensitivity, precision and accuracy is necessary in order to determine the quality of clinical grade DNA intended for therapeutic use. Plasmids purified from Escherichia coli fermentation exist predominantly in the supercoiled form (SC) the other two topoisomers present in the final product are mostly the open circular (OC) and linear forms . Different chromatographic conditions were tested and the separation was optimized in terms of buffer and pH selection as well as in terms of gradient slope and column length. The results were compared to the results obtained with established analytical methods.
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.
A number of IgM monoclonal antibodies are currently in development for treatment of autoimmune disease, infectious disease, and cancer. Growing interest in these molecules has created a need for an accurate, rapid, simple analytical method to measure IgM levels in cell culture supernatants, and to document the distribution of IgM and protein contaminants in chromatography fractions. High performance protein A columns are used for this application with IgG monoclonals, but IgMs are easily denatured by the harsh conditions required for elution of most affinity ligands. However, IgM monoclonals often exhibit strong retention on either cation exchangers, or anion exchangers, or both, making ion exchange chromatography a potential candidate for this application.
The large size of IgMs makes them a major challenge to particle-based chromatography media. Pentameric IgM has a mass of about 0.96 Md, and hexameric IgM about 1.15 Md. Their diffusion constants are about 2.5 x10-7 cm2/sec, about twice as slow as IgG. Since particle-based chromatography media mostly rely on diffusion for mass transport, both resolution and capacity are im- Figure 4 illustrates a modified anion exchange gradient configuration for monitoring the amount of IgM expressed in cell culture supernatants. A wash step was introduced to better remove con- paired, and increasingly so at higher flow rates.
Monolithic ion exchangers are characterized by an interconnected system of channels with diameters ranging 0.5 to 2.0 microns. This pore architecture supports convective flow, which conserves high resolution at high flow rates. The lack of a void volume removes the major source of dispersion in chromatographic systems. This contributes to sharper peaks, which improves both resolution and sensitivity. Capacity is also conserved at high flow rates. This permits use of a microcolumn format that minimizes assay time and buffer consumption. This combination of features should make monoliths effective analytical tools for IgM.
Membrane based anion exchangers are being used increasingly for purification of monoclonal antibodies. The transition from particle-based anion exchangers is driven partly by the convenience of membranes and partly by the cost saving associated with their disposability, however the feature that makes them functionally superior is more effective mass transport.
IgM can be used for several purposes such as early detection of certain diseases or, when labelled, localized cancer tumours. For their purification commonly chromatography is used. Methods for purifying such big molecules (M.w. around 950 kDa) are usually long and time consuming since these molecules have extremely low mobility therefore mass transfer between mobile and stationary phases is significantly reduced. When purified using affinity mode, serious decrease in IgM activity can occur because of long exposure to low pH in which they are unstable. Furthermore, because of their size, the IgM capacity of convenctional resins is rather low. CIM monoliths were already successfully used for fast separation of large molecules. In this work we tested applicability of anion-exchange CIM monolithic columns for preparation of IgM.
Gene therapy has already shown some great results in treatment and cure of some monogene diseases, such as diabetes. While the use of genetically modified viruses raises safety concerns, synthetic formulations of genes inserted in plasmids are regarded as safer. At present, most clinical trials involve plasmids smaller than 10 kb. However, the concern that regulation of the functioning of the gene is ensured together with the expectation of the progression of gene therapy to multigene disfunctions, like cancer or complex nevrodegenerative disfunctions (Alzheimer disease), will require the production of larger plasmids .
Commercially available CIM® disk monolithic columns are intended for very fast analyzes and laboratory purification. Their shape is a compromise to achieve acceptable resolution and binding capacity what make them suitable for wide range of laboratory applications. Separations of complex protein mixtures can be carried out within just a few seconds because of flow unaffected resolution and, on the other hand, purification can be effectuated with high productivity due to flow-unaffected dynamic binding capacity . However, in many cases in the field of molecular biology, only a limited amount of sample is available. In such a case it is beneficial to work with small columns having high resolution or they can be used as affinity columns or bioreactors saving significant amount of valuable ligand. Having this goal in mind we developed CIM® disks with the volume of 1/10th and 1/100th of original volume. In comparison to conventional CIM® disks, they exhibit higher resolution and lower limit of detection, therefore smaller concentrations of target macromolecules can be detected. The separation ability and the protein capacity were tested on anion and cation exchange 3.4 mL and 34m L mini disk monolithic columns.
Analysis of a large numbers of samples requires chromatographic supports that not only enable fast separation and purification of a target biomolecules from a complex matrix but are also involved in an automation process. The 96 – microtiter plate format enables both. Although they are routinely used for decade's only recently few reports about the microtiter plates bearing monoliths as a separation media, were reported . Because of advantageous properties such as flow unaffected dynamic binding capacity and resolution 96 - microtiter plates with methacrylate based monolith were prepared. Characterisation of such plate demonstrated that uniform flow rate can be achieved through all wells and no leakage is present. Efficient separation of proteins was achieved within minute. Furthermore CLC (Conjoined Liquid Chromatography) concept  originally derived for analytical columns on CIM disk, can easily be extrapolated to microtiter plates. We demonstrated that multidimensional chromatography with 96 – well plate is feasible and can further accelerate screening processes.
The Inter-alpha inhibitor protein family is comprised of complex plasma proteins that consist of a combination of multiple polypeptide chains (light and heavy chains) covalently linked by a chondroitin sulfate chain. The major forms found in human plasma in high concentration are Inter-alpha inhibitor (Ial), which consists of two heavy chains (Hl & H2) and a single light chain, and Pre-alpha Inhibitor (Pal), which consists of one heavy (H3) and one light chain (Fig 1). The light chain (bikunin) is known to inhibit several serine proteases, such as trypsin, human leukocyte chistase, plasmin and cathepsin G which are involved in inflammation, sepsis, tumor invasion and formation of metastasis. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAli 6931) was developed in our laboratory.
The analysis of molecular interactions is a key part of the drug discovery process, and analytical techniques are available for studying in vitro the ligand/target complex since the early stage of the drug development process.
With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications.
Human recombinant acetylcholinesterase (hAChE) represents a well-known target for drug-discovery in Alzheimer’s Disease.
The rapidly growing interest in the area of proteomics induces intensive efforts to find robust, automated and sensitive high-throughput analytical tools. In this context, the concept of solid-phase digestion (ex. trypsin immobilization on a solid support) has received great attention in the last years. Trypsin (EC 22.214.171.124) has been covalently immobilized on different monolithic supports and resulting bioreactors used as immobilized enzyme reactors (IMERs) for on-line digestion, peptide separation and peptide mapping. Bioreactors efficiencies were evaluated with different recombinant proteins after on-line digestion. The technique used for the separation and identification of peptides was high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS).