AAV vector lots are generally a heterogeneous mixture of empty particles (i e do not contain DNA) and full particles (i.e. contain DNA). Different spectrometric based methods can be used to establish the ratio between full and empty AAV particles, but accurate evaluation of empty/full ratio is often obstructed due to complex spectroscopic behavior of empty and full AAV particles, such as poor separation and impurity overlapping. An approach that takes difference in physical chemical properties between empty and full capsids into account overcomes limitations of spectrometric based evaluation of empty and full AAV particle ratio.
Chromatographic separation of empty and full AAV 2 8 capsids was achieved on the CIMac AAV full/empty analytical column (strong anion exchanger, QA quaternary amine chemistry) with the PATfix™ system using a linear NaCl gradient at pH 9.0 Signal response from three different detectors connected in series was analyzed fluorescence (excitation 280 nm emission 348 nm), light scattering 90 angle, LS) and UV absorbance 260 nm and 280 nm).
Serotype 10 adeno-associated virus (AAVrh_10mCherry) was analysed on the PATfix™ 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).
Immunoaffinity columns using antibodies as ligands against mammalian proteins could be used for different applications in protein expression control and, if a standard available, for direct protein quantification in complex sample solutions. Additionally, these columns are ideal for polishing step of recombinant proteins, such as mammalian receptor Fc fusion proteins. Most importantly, such columns could extract a significant amount of a single membrane protein from native source, suitable for downstream analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against RAE-1 GPI anchored glycoprotein were developed (CIMmic HDZ - @RAE-1 column) as a part of Glycomet project with the main goal to analyze the antigen glycoprofile.
Hydrazide-activated (HDZ) columns were proven to be a product of choice for making the most effective immunoaffinity columns. They take advantage of a special hydrazide linkage that binds antibodies through the carbohydrate residues on their Fc regions. This leaves the antigen-binding domains fully accessible to enable the most effective capture of desired target (Figure bellow).
CIMac™ HDZ monoliths make HDZ-immobilized antibody columns even more effective. Because of their large channel size and the efficiency of convective mass transport, they eliminate the long loading residence times that are required for affinity chromatography on porous particle columns. Flow rates of 5–10 column volumes per minute allow complete purifications in a few minutes, even when the source material contains a low concentration of antigen. The same performance is achieved whether a small peptide or a large bio-assemblage like a virus particle or extracellular vesicle is isolated. The combination of HDZ monoliths and the immobilization protocol offers a strong tool for fast antigen isolation from complex biological sample (plasma, lysate, etc.) and consequently sensitive antigen quantification. An example of CIMac™ HDZ application is a purification of fibrinogen from human plasma.
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.
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.
The upstream and downstream monoclonal antibody (mAb) bioprocessing makes them susceptible to physical and chemical modifications. In the biotechnological production process of mAbs, structural variations may arise due to some enzymatic activity. Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity and cation-exchange chromatography (CEX) is one of the typical approaches for mAb charge variant analyses. We tested several CEX columns under different conditions and the best column for isotype separation was weak cation-exchanging CIMac COOH chromatographic monolith in pH gradient. We have proven a flow independent separation of mAb charge variants and in this way, a resolution comparable to classical CEX particulate-based analytical columns was achieved in only 6 min analysis time.
CIMac™ r-Protein A Analytical Column is short bed, high performance monolithic column . Primarly is intended for fast, efficient, and reproducible qualitative and quantitative analyses of Immunoglobulin G (IgG). It is suitable for use with HPLC and UPLC systems. Quantification of Immunoglobulin G is possible between 0.2 μg and 20 μg. Its small volume and short column length allow operation at high volumetric flow rates ( up to 3mL/min). The information about product quantity and purity is thus generated in just 1 minute! The column has innovative symmetric design for bi-directional flow, also extending column lifetime.
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.
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.
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.