On May 12th, the biaseparations.com website will be retired and migrated tosartorius.com.Learn moreabout our combined offering today!
2012

Potato spindle tuber viroid (PSTVd) is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and uncapsidated RNA molecule with 359 nucleotides and no coding capacity. Because of its complex secondary/tertiary structure it is very stable ex vivo and it is easily transmitted mechanically by contaminated hands, tools, machinery, etc. In this work, we describe the development and optimization of a method for concentrating PSTVd using CIM monolithic supports.

Attachments

Full view

Objective – Influenza VLP
• Complex structure
• Different protein components
• Host cell derived lipid membrane
• ESAT6 epitope of M. tuberculosis engineered into influenza hemagglutinin [1,2]
• Optimal vaccine candidates
• Induce strong immune response [3]
• Contain no genetic information

Attachments

Full view

In the last few years pharmacology has made a big step towards the new type of drugs, called biological drugs. Popularity and market for biological drugs grew exponentially, so did the need for fast and inexpensive purification. Classic liquid chromatography columns were unable to separate biological compounds in industrial quantities, therefore the scientists were looking for alternatives. One of them are monolithic materials. Monolithic materials, especially methacrylate monoliths, are becoming more and more popular in separation processes due to their fast separations, low pressure drop and mechanical stability.

In the context of preparing new columns and improving existing ones, we need to know every single chemical as well as mechanical property of our monolithic material. Here we present some key data and interesting correlations between mechanical and structural properties of GMA-co-EDMA porous monolith. In the first paragraph we compare nonmodified and DEAE modified monoliths with different average pore size and porosity, regarding to their compression and tension properties. The second paragraph deals with the impact of these parameters on the permeability of the column during separation.

Attachments

Full view

Commonly, epoxide-based monoliths used as porous supports in affinity chromatography are synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) by free radical polymerization.

We prepared an epoxide-based monolith by self polymerization of polyglycidyl ethers where the epoxy groups serve as functional groups for the polymerization reaction as well as for the immobilization of the ligand.

Attachments

Full view

Monolith technology has been employed in chromatography for a variety of applications using diverse substrates. The development of different column chemistries has led to the Thermo Scientific ProSwift line of monolith columns for analytical protein separation by ion exchange and reversed phase. Separation of biomolecules can be achieved at elevated linear velocities with minimal loss of resolution. Columns are designed to withstand extreme pH cleaning, desired for sterilization. The backbone and functionalization are optimized for high mass loading for small-scale preparative applications, the ideal first dimension separation of crude biological samples. Combined with increased sensitivity of a 1 mm format, detection of proteins of very low copy number in a crude samples is achievable.

We discuss here the ability to produce highly-reproducible columns with excellent stability as well as characteristics required for fast small-scale preparative analysis. HPLC column selection is a challenging task, specifically where the mixture contents is somewhat unknown. Many factors influence the choice of column used; chemistry, robustness, and reproducibility. For quality assurance, columns should be chosen that are reproducible both run to run and batch to batch. To prevent cross-contamination between samples, carryover and sterilization should be considered. For semi-prep, a combination of high mass loading and good resolution enable increased purity of peak fractions. Format and operational flow rate should be considered with respect to multidimensional analysis.

Attachments

Full view

2011

Over the last two decades,the potential of virus-based biopharmaceuticals for application in gene therapy and vaccination brought new challenges in bioprocess development. Particularly, the downstream processing (DSP) of enveloped viruses shifted from bench-scale towards robust, scalable and cost-effective strategies to produce clinical grade viralvectors. Lenti viralvectors(LVs) hold great potential in gene therapy due to their ability to transduce non dividing cells and their capacity to sustain long-term transgene expression in several target cells, invitro and invivo1. However, despite significant progress, the quality of LV preparations, the purification and the concentration of high titers of these vectors is still cumbersome and costly. In this work, disposable membrane technologies, involving microfiltration, anion-exchange chromatography (AEXc) and a final ultrafiltration step, were the basis for the development of an optimized purification process for LV.

Attachments

Full view

2010

Application of plasmid DNA for gene therapy and vaccination has gained huge interest in last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Major influence on achieving regulatory demands in pDNA production has downstream processing and in order to get optimal purity different purification techniques have to be included. 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 enabled excelent purity and productivity.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

Analysis of a large number of samples requires chromatographic support that not only enables fast separation and purification of a target biomolecule from a complex matrix but also support an automation of a process. The methacrylate 96-well monolithic plate format enables both. 96-well monolithic plate reduces experimental time because it allows fast and efficient evaluation of parameters for binding and elution conditions. This format is a quicker alternative to several consecutive tests on chromatographic column.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

2009

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.

Attachments

Full view

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%.

Attachments

Full view

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.

Attachments

Full view

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) [1]. 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.

Attachments

Full view

2008

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 [1].

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 [2]. 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.

Attachments

Full view