Biological samples often consist of a main component, such as albumin in serum, and many other constituents, present in smaller quantities, but nevertheless of high importance in biological systems. When detection of the low-abundance molecules is needed, the main component could interfere with the analyte, complicating the analysis or even making it impossible. In such cases a possible approach is to remove the interfering main component from the sample before the analysis.
Monolithic columns (CIM®) are a great foundation to build affinity chromatography methods, as they offer fast flow rates and can be modified to accomodate various ligands. We selected two most promising approaches for oriented binding of antibodies to the monolithic support. One approach was to bind antibodies to a protein A (pA) column with consequent crosslinking of the protein complex. The other approach was to chemically activate antibodies and bind them selectively to hydrazide-modified (HDZ) monolith surface.
Exosomes are nano-sized vesicles that are released by many different cell types. They are involved in the transport of a wide range of signalling molecules, including mRNA, microRNA and proteins. Exosomes have been found into body fluids and multiple roles have been ascribed to exosomes, in particular in cell signalling where it has been demonstrated their correlation to disease progression and their overexpression as specific tumour cell biomarkers, suggesting their important role in their diagnosis.
This initial screening oriented towards the separation of exosomes from a cell culture supernatant, has been developed by BIA Separations in collaboration with Exosomics Siena. Exosomes used for this study were cultivated in two different cell lines, MeWo and LNCap, and, after the harvesting, a relatively pure target molecule was obtained after several centrifugations, filtrations and batch affinity capture step with a commercial purification kit. In order to speed-up the process and bring current DSP on a higher level, a novel purification approach based on chromatography, using CIM® monolithic columns was investigated. Monolithic supports represent a new generation of chromatographic media. Due to their large inner channel diameters and enhanced mass transfer characteristics, methacrylate monoliths offer efficient and fast separation of large biomolecules like vescicles, pDNA, viruses and monoclonal antibodies. High binding capacity, good product recovery and resolution are also benefits of monoliths. Different samples, (Standard batch purified exosomes, Culture supernatant filtered, Culture supernatant non-filtered), derived from MeWo and LNCap culture media,, were screened. QA, SO3, DEAE and OH CIM 1mL tube - 6μm pore size were screened. CIM® QA - 6μm pores was chosen.
Interactions between antibodies and their antigens are highly selective and therefore immensely popular for affinity chromatography. Consequently, numereous antibody immobilizations were performed on monolithic supports via different activation chemistries in the last decade. Despite the work already done there was no systematic study, where as many as possible activation chemistries were tested for the immobilization of a model monoclonal antibody with subsequent chromatographic characterization of the affinity support. In this work, various preactivated CIM monolithic columns were used for the immobilization of a model monoclonal IgG.
Monolithic ion exchange CIM® (Convective Interaction Media) columns have been proven in quantitative analysis of different immunoglobulins such as IgM and IgG from human plasma or cell supernatants. The separation mechanism is based on ionic interactions between the ion exchange monolith and immunoglobulin that are controlled by salt concentration. Here we present another possibility of IgM determination based on monolithic CIM® OH columns where the interactions may be controlled by changes in salt concentration or by pH increase. A method for quantitative HPLC determination of IgM in cell supernatant with fluorimetric detection was developed on CIM® OH column (0.34 mL) by means of pH increase. Optimal separation of IgM from cell supernatant matrix was achieved by combining acetate and phosphate buffer in a suitable gradient profile. Two different quantification methods, i.e. calibration curve and standard addition.
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.
Enrichment of phosphopeptides prior to LC-MS analysis is a crucial sample preparation step because of their low stoichiometry in biological sample, longer retention on reversed phase columns, and lower ionization efficiency compared to non-phosphorylated peptides .The use of metal oxides, most prominently of TiO2 enabled efficient and relatively simple phosphopeptide-enrichment. In this study a new monolithic column from BIA Separations containing immobilized TiO2-nanoparticles was tested for its ability to enrich phosphopeptides. The TiO2-column was also tested for possible carryover originating from biological samples. In conclusion, tested monolithic TiO2 columns show significant binding ability for phosphopeptides and are considered as suitable for phosphopeptide enrichment.
The demand for human immunoglobulin is invariably increasing on an annual basis. To satisfy demands, different manufacturing processes are used to isolate immunoglobulins from human plasma. A quest for alternative paths in manufacturing not only requires development of the most economical manufacturing process, but also a rapid method development and development of reliable analytics for manufacturing monitoring. For an efficient improvement of the purification methods as well as for in-process control during manufacturing stage, the usage of reliable and fast analytical techniques are of crucial importance.
Fast and reliable fingerprint-based method for characterization of immunoglobulin G (IgG) prepared from Cohn I+II+III paste in two chromatographic steps is presented. The fingerprint method bases on partial separation of proteins in linear gradient on CIMac QA 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples during the IgG production from Cohn I + II + III paste, however, a very accurate qualitative information about the composition of the sample can be obtained in less than 5 minutes.
In recent years bacteriophages were identified as a useful potential tool for different applications such as alternative to antibiotics, detection of pathogenic bacteria, delivery vehicles for protein and DNA vaccines and as gene therapy delivery vehicles. 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 CsCl2 density gradient ultracentrifugation, which is however difficult to be scaled-up. An alternative is chromatography, which already proved to be efficient for separation and purification of certain virus types. Methacrylate monoliths (CIM Convective Interaction Media® monolithic columns) were designed for purification of bionanoparticles and they already proved to be very efficient for concentration and purification of several plant and human viruses (influenza A, influenza B, adenovirus type 5, hepatitis A and others).
Our aim was to investigate whether CIM methacrylate monolithic columns can be implemented for purification of phages. Staphylococcus aureus phage VDX-10 was selected. Chromatographic support chemistry and buffer screening led to development of purification method on strong anion exchanger. Optimised single step purification method developed for S. aureus VDX-10 phage on CIM® QA monolithic column resulted in efficient removal of host cell DNA and proteins with high recovery of viable phage.
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
The development of safe, effective, and affordable vaccines has become a global effort due to its vast impact on overall world health conditions. A brief overview of cancer vaccine characterization techniques, especially in the area of high-resolution mass spectrometry, is presented. It is highly conceivable that the proper use of advanced technologies such mass spectrometry, along with the appropriate chemical and physical property evaluations, will yield tremendous in-depth scientific understanding for the characterization of vaccines in various stages of the development. This work presents the physiochemical and biological characterization of two cancer vaccines: Racotumomab and Her1-ECD. Racotumomab monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. The Her1-ECD is a vaccine preparation based on the extracellular domain of HER1 and it is being evaluated in Phase I clinical study in patients with refractory prostate cancer.
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.
Glycosylation is one of nature mechanism for invreasing the diversity of protein structures affecting biophysical vjaracterostocs and bioactivity. Glycoproteins exist as mixture of different isoforms ("glycoforms"). In this mixture a group od different glyco components is attached to individual glycosylation site. Different glyco componets attached to the same site may have diggerent effect on biophysical charachteristics of glycoproteins. The type of glycosylation and the degree of heterogenity are important for many reasons starting from stability, activity, clinical efficency (toxicity, pharmacokinetics, immunogenicity), to standardization and patentability.
Thus, it is necessary to separate glycoforms and as much as possible to difine the heterogenity i.e. population of of glyco components attached to the singele glycosysilation site.
External invertase is a widely usef model for studying the influance of the glyco-component on protein stability. External invertase from yeast Sccharomyces cerevisiae has 14 potential N-glycosylation sites in the sequence, 13 of which are fully or partially glycosylated with olygomannans of varying sizes.
Extensive research in the last two decades has led to the realization of Immunoglobulin M (IgM) as a potential therapeutic and diagnostic agent for autoimmune diseases, infectious diseases and as an AIDS and cancer vaccine. Growing interest in these molecules has created a need for an accurate, rapid and simple analytical method to measure IgM concentrations during the production (in-process control) in cell culture supernatants as well as in all purification steps in the downstream processing.
Convective interaction media (CIM) monolithic columns has been increasingly recognized as a quantification tool for large molecules. Affinity ligands like protein A and protein G are the most common ligands used for antibody capture and analysis.
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
There is an increasing demand for highly purified immunoglobulin G since they have found wide range of potential application in immunodiagnostics and immunotherapy.
Human IgG (hIgG) consists of four subclasses (IgG1, IgG2, IgG3 and IgG4) that show differences in some of their physicochemical characterictics and biological properties.
The present research project aims to separate subclasses of hIgG using monolithic stationary phase by SMB technology.
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.