Serotype 10 adeno-associated virus (AAVrh_10mCherry) was analysed on the PATfix™ HPLC system with the CIMac™ AAV full/empty analytical column to estimate the ratio of empty and full AAV particles based on the peak area of the chromatogram given with three different detectors. AAV included a protein capsid containing single stranded DNA. CIMac™ AAV column consisted of a strong anion exchanger with QA chemistry (quaternary amine).
Poster was prepared by Blaz Goricar and presented at ISBioTech 9th Spring Meeting where it was awarded the first prize. Congratulations!
This poster presents fully scalable non-affinity purification strategy that has been proven to be effective for all AAV serotype tested to date. Cell lysate is directly subjected to column purification after removal of cell debris without requiring a concentration step using tangential flow filtration. The process consists of three chromatographic steps. Hydrophobic interaction chromatography on a CIMmultus OH monolith is used for initial virus capture and purification. Precipitating salts are used at 1.0–2.0 M to achieve virus binding. Most of the small molecule contaminants and proteins are eliminated in the flow-through. AAV co-elutes with a highly reduced population of contaminating proteins. DNA-protein complexes are very strongly retained and require NaOH for removal. Intermediate polishing is performed with a CIMmultus SO3 cation exchange monolith. The AAV fraction from the capture step is titrated to a pH value of 3.5—5.0 and diluted to binding conditions. Sugars and surfactants are added to suppress non-specific interactions with tubing and containers, and the product is eluted in a salt gradient. Final polishing is conducted on a CIMmultus QA anion exchange monolith which separates empty capsids from full capsids. This is achieved in a salt gradient at alkaline pH. For more information please refer to BIA Application note A048 (www.biaseparations.com/applications).
Chromatography is a useful purification method for large biomolecules and virus manufacturing and it is easily scalable to large production volumes. Convective Interaction Media (CIM) monolithic columns constitute of large flow-through channels and consequently have high surface accessibility of binding sites. Preferences of CIM monolithic columns are flow independent performance, resulting in fast separation, concentration, purification, impurities removal, and analytics of biopharmaceuticals.
The aim of the study was to develop Influenza virus purification platform, which can be used for several virus strains. The main objective was to develop a process with as little as possible of intermediate steps, especially omitting Tangential Flow Filtration (TFF) or other sample pre-treatments with high host-cell DNA and protein removal, as well as to achieve high binding capacity of the Influenza virus per mL of monolithic support.
During recombinant adeno associated virus (rAAV) downstream processing, a large amount of host-cell and product related impurities needs to be removed from the product. Succesful process on laboratory scale, such as Cesium chloride purification, lacks scalability when the process is due to be transfered to larger industrial scale. The aim of the study was to develop robust, fast and effective rAAV virus purification platform, which can be used for several AAV serotypes with various inserts. Lysed harvest and supernatant of rAAV9 were first captured and concentrated on CIMmultus™ OH column, followed by intermediate step on CIMmultus™ SO3 column and further polishing on CIMmultus™ QA column. Derived purity of industrial scale monolith purification product was compared to laboratory scale purification.
CIM® chromatographic monoliths enable high 1) productivity of pDNA downstream process (DSP) due to high dynamic binding capacity for pDNA in small elution volumes and short chromatographic runs; 2) high resolution power due to convective-based mass transfer.
Sample displacement mode utilizes different relative binding affinities of components in a sample mixture and separates pDNA isoforms under overloading conditions - where sc pDNA isoform acts as a displacer of oc or linear pDNA isoform.
Preparative scale chromatographic separation of open-circular (oc) from supercoiled (sc) plasmid DNA (pDNA) isoforms has been already established on CIM® C4 with high ligand density (C4 HLD) monolithic columns with sample loading in 3.0 M ammonium sulphate (AS). The process requires high molarity of AS, increasing the overall cost of the process. Sample displacement chromatography (SDC) can be used as an alternative to decrease the AS concentration required during loading onto hydrophobic chromatographic supports. This study compares three chromatographic monoliths with different hydrophobic ligands on the surface (C4 HLD, pyridine and histamine) for the purification of different pDNA vectors in SD mode.
New vaccines against Influenza A are required each year to keep up with the most virulent evolving strains. This highlights a need for predictive analytical tools that can aid purification process development and validation. Rapid and reliable quantification of Influenza A virus is therefore of the utmost importance for enabling good yields and controlling the costs of the downstream processing. Here we demonstrate the ability of monolithic chromatography media to produce process predictive profiles that can document ability to remove impurities and obtain high product recoveries.
CIMac™ Analytical Columns are short bed high performance monolithic columns offering all the advantages of CIM® monolithic technology. Their small volume and short column length allow the operation at high volumetric flow rates enabling to receive the information about the product quantity and purity in just a few minutes. Hence, the CIMac™ Analytical Columns can be effectively used for the in-process and final control of various samples from different purification process steps.
Adeno-associated virus (AAV) vectors of various serotypes are considered to have high potential for gene therapy applications. Currently, manufacturing of AAV vectors faces the challenge of co-production of incompletely formed particles lacking a recombinant viral genome. Empty capsids increase the dose of total AAV administered for efficient transduction and are thought to cause unwanted immunological reactions against the virus.Removal of empty capsids during manufacturing, as well as analysis of empty/full AAV particle content is therefore a critical requirement for any AAV production process. This poster demonstrates how CIMmultus™ QA monolithic columns can be used to remove empty AAV capsids from the product chromatographically in a single step.
Since plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy, one or more chromatographic steps are often used in the downstream processing train. High ligand density butyl-modified (C4 HLD) monolithic support is currently used in a polishing step of a pDNA purification process (1) and is mainly focused to supercoiled (sc) pDNA isoform separation from the open circular (oc) and linear pDNA isoform as well as for removal of remaining gDNA and RNA. The goal of the study was to compare the productivities of two variations of the polishing chromatographic process employing monoliths – classical bind-elute (BE) versus recently described (2) sample displacement purification (SDP). Classical purification requires high concentration of ammonium sulphate (AS) during loading step and elution is then achieved by descending AS gradient. SDP utilises different relative binding affinities of components in a sample mixture and separates pDNA isoforms under overloading conditions, where sc pDNA isoform acts as a displacer of oc or linear pDNA isoform.
One of the major requirements for pharmaceutical-grade pDNA is its high homogeneity, being mostly in supercoiled (sc) isoform. Chromatographic separation of sc pDNA from open coiled (oc) or linear isoform is challenging due to their similar interactions with the chromatographic phases. Promising separation efficiency of pDNA isoforms was proven on recently developed histamine modified monolithic chromatographic column in descending ammonium sulfate gradient. The aim of the study was to further optimise the chromatographic conditions for sample analysis, where all three isoforms would be baseline separated.
Determining the concentration of viruses is a crucial step in any production process. The most commonly used methods for virus quantification are either based on the infectivity of the virus (plaque assay, TCID50) determination of their genomic material (qPCR), or protein content (SRID, ELISA) and are very cumbersome and time consuming. HPLC analytical methods represent a fast alternative to these assays since they provide information on the virus content and purity in a matter of minutes. Due to the structural properties of the monolithic supports, monolithic analytical columns offer a great advantage over particle based HPLC columns in terms of time and their ability to separate large biomolecules, like viruses, VLPs, pDNA.
In this poster the performance of the CIMac™ Adeno Analytical Column – a monolith based anion exchange column, designed for fast and reproducible analyses of adenoviruses was evaluated. CIMac Adeno column can be used for designing a fast finger printing method that is applicable for monitoring the DSP production process of adenoviruses. Once the basic analytical parameters like linearity and sensitivity are determined using a purified adenoviral standard, the metod can be applied for quantitative determination of adenoviruses.
The challenge of efficient purification of gene therapy vectors
• The most commonly used gene transfer vectors are adenoviruses, lentiviruses, adeno-associated viruses, retroviruses, vaccinia viruses, and pDNA
• Due to their large size and sensitivity to pH, temperature and shear stress, purification is challenging and time-consuming
• A fast and efficient downstream processing purification method is required to isolate sufficient amounts of vectors with the final purity and state that conforms to stringent regulatory demands.
Solution: Convective Interaction Media Monoliths
• Convective interaction media (CIM) monolith chromatography
• Functionalised polydimethacrylate (QA, DEAE, OH, SO3)
• Precisely defined pore sizes
• Radial flow of solute
• Convective mass transfer
A monolith is a stationary phase made of single piece of porous material. Unlike conventional particle-shaped chromatographic supports, the pores of the monolith are interconnected and form a network of channels with diameters ranging around 1500 nm. The binding sites in these channels are highly accessible for target molecules and since the predominant mass transfer depends on convection rather than diffusion, the dynamic binding capacity is flow independent. These characteristics make the monolithic supports suitable for fast separation and purification of large biomolecules such as proteins, DNA and viruses, which sometimes exceed 200 nm in size and thus have low diffusion constants.
In this work we tried to quantify influenza A virus using an analytical CIM monolith column. First a screening of available CIM stationary phases was performed in order to establish the optimal stationary phase for the binding of the virus. The effect of the mobile phase composition and pH on the recovery and peak shape of the virus was investigated. Linearity was examined. The amount of virus in the flow-through and elution fractions was determined with the haemagglutination assay and the purity of the fractions with SDS PAGE. All experiments were performed with an inactivated Influenza A/Wisconsin PZC whole virus sample that was produced in eggs.
Monolith chromatography media coupled with metal affinity ligands proved superior to the conventional particle-based matrix as a plasmid DNA (pDNA) purification platform. By harnessing the differential affinity of pDNA, RNA. Host cell proteins and endotoxin to copper ions in the solution a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl2-induced precipitation. RNA and remaining endotoxin were subsequently processed by copper immobilized metal affinity column employing either monolith or particle-based matrix where both RNA and endotoxin were removed below detection limit with almost complete recovery of pDNA in the monolith was found to have several advantages in terms of handling feedstocks crowded with RNA in a concentration-independent manner and exhibiting flowrate-independent dynamic binding capacity for RNA. This enabled monolith-based process to be conducted at high feed concentration and flow rate. Resulting in pDNA vaccine purification at a high yield and purity and the process conditions investigated, the use of monolith column gave at least three fold higher productivity for recovery of purified pDNA as compared to the particle- based column, demonstrating its potential as a more rapid and economical platform for pDNA vaccine purification.
The present study describes a new methodology to quantify and monitor the quality of supercoiled (sc) plasmid DHA (pDLIA), using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows distinguishing the plasmid isoforms by a NaCl stepwise gradient. The selectivity, Linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a sc plasmid concentration up to 200 µg/mL. The main advance achieved by using this monolithic method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, in a 4 minutes experiment. This work also intends to evaluate the possibility to assess the sc pDNA present in more complex samples, allowing the control of the samples recovered from different bioprocess steps.
Recombinant Adenovirus (rAd) is commonly used for vaccination and gene transfer for cancer applications. This vector is widely used in phase I/II clinical trials. Therefore we believe that upstream and downstream processes should be improved.
We developed a production manufacturing process for rAd serotype 5 n HEK293 grown into disposable fixed-bed iCELLis™ bioreactors (ATMI LifeSciences). The purification process was reduced to one single chromatography step using the Convective Interaction Media, anion exchanger (CIM ® QA monolithic column, Bia Separations).
Briefly, rAd particles were extracted from cells using Triton X-100, depth filtered to discard cell debris, captured and purified out on CIM ® QA. The shallow gradient used for the elution of the vector allowed the separation of different rAd particles populations more or less enriched in full particles. A final step based on Tangential Flow Filtration (TFF) in hollow fibers allowed the removal of remaining impurities and the formulation of the vector batch.
In addition, we developed an analytical method on CIMac™ QA analytical column (Bia Separations) to characterize the different steps of the process, and to track the differences linked to the production runs to increase the robustness of the process. This method provided elution profiles for each step as well as titer of the purified rAd in the final step.
The rAd was produced in an iCELLis™ nano fixed-bed bioreactor (0.5-5.3 m2), purified in a 8mL CIM ® QA monolithic column, scaled up in a medium-scale size 80mL column. We are currently extending the rAd production in a 133m2 iCELLis I000™ bioreactor with a purification step using a 8L CIM® QA monolithic column to purify out up to 1x1015 vector particles.
Monolithic supports represent a new generation of chromatographic media. Due to their large inner channel diameters and enhanced mass transfer characteristics, methacrylate monoliths (CIM® monolithic columns) offer efficient and fast separation of large biomolecules like pDNA, viruses and monoclonal antibodies. High binding capacity for viral particles, good product recovery and resolution are also benefits of monoliths. During loading of MDCK cell-derived H1N1 inactivated influenza virus particles onto monolithic columns, increased back pressure is sometimes observed. This is especially an issue if a large amount of virus needs to be purified since the back pressure depends on the loading volume. The goal of this work was to determine the factors contributing to this effect. We tried to prevent the increased back pressure by treating virus harvests with different precolumn phases (LRATM - Lipid removal agent, Amberlite® XAD 7HP, epoxy monolithic column) and by filtering the virus material before loading it onto the column. To compare different pre-treatment strategies of the virus material the dynamic binding capacity of CIMac QA for virus was first determined, resulting in approximately 1x1013 virus particles per ml. Than loadings of the pre-treated virus material at 75% of the column capacity were performed and mass balances for the virus, DNA and proteins were investigated. Another goal of this work was to find a good regeneration strategy for the columns where increased back pressure occurred. For this reason different regeneration procedures using lipase, benzonase, 2-propanol and NaOH treatment were tested on the columns with increased back pressure.
Traditional waste water treatment usually does not remove or inactivate all of the potentially pathogen microorganisms present in the waste water. This is especially true for enteric viruses that are introduced into the environment through the discharge of effluent from waste water treatment plants - WWTP (Simmons et al, 2011). Although discharged concentrations of viruses are low they can still lead to infection. For some enteric viruses ingestion of only 10 - 100 virus particles is enough to initiate the disease, what calls for very sensitive detection methods. It has been previously shown that CIM-quaternary amine (QA) monolithic supports are a good tool for concentration of viruses in water (Gutierrez-Aguirre et al, 2011). Here we go one step further and evaluate CIM monoliths not just for concentration of enteric viruses but also for their removal from effluent waters.
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.
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 
• Contain no genetic information