Extracellular vesicles (EV) are lipid bound products secreted by cells. Among them, exosomes have great potential for clinical applications. Animal and human-derived components used in cell culture, such as fetal bovine serum (FBS), naturally contain exosomes that can cross-contaminate the desired product. In order to study exosomes derived from cells of interest, multiple producers have come up with exosome-depleted FBS (EV (-) FBS) generated using different approaches. In this work we evaluated commercially available EV (-) FBS supplements for residual exosome content and tested their performance in upstream exosome production process. The analysis was performed with PATfix high pressure liquid chromatography (HPLC) system using PATfix size exclusion (SEC) analytical method.
Removal of host cell DNA is essential for all human-injectable biologics. This poster shows a method for achieving low host cell levels in preparations of exosomes. Purified exosome samples were prepared with anion exchange chromatography (AEC) and pre-treated with tangetial flow filtration (TFF) and nuclease treatment. Results are compared with an experimental control using TFF and size exclusion chromatohraphy (SEC).
The steps in purification process are illustrated by analytical size exclusion chromatography (SEC) on PATfix HPLC system with in-line UV, MALS and fluorescence detectors and by staining with Picogreen reagent. This technique visualizes sample composition by size, UV, light scattering and fluorescent properties.
Exosomes fulfill a critical role as communicators among cells, with targeting and message content depending on their surface receptors and payload. This makes them obvious candidates for an extensive range of diagnostic, therapeutic applications and a need for a fast, robust and scalable purification procedure.
CIMmultus™ monolithic columns are designed to meet the special fractionation needs of very large biologics like exosomes.
We show examples of exosome purification from cell culture with CORNERSTONE Exosome Process Development Pack and analysis of exosomal vesicle populations by Image stream flow cytometry.
This poster shows how Multi-Angle Light Scattering detector and Fluorescence detector couppled to PATfix analytical HPLC system can be used to track extracellular vesicles through purification process. Samples were analyzed by analytical size exclusion chromatography (SEC). On SEC cell culture components diffuze into pores of chromatographic media and are separated (mostly) based on size. Particles larger than the media pore size are excluded in the void peak. This peak represents extracellular vesicles including apoptosomes, microvesicles and exosomes as well as cell debris and aggregates.
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.
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.
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.
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 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.
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.
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.
Protein L binds certain types of kappa light chains containing Fv and Fab fragments prepared from antibodies. In the case of IgG's the strong binding affinity refers only to human, mouse and rat species. It offers an advantage over Protein A and G as it binds to kappa light chains regardless of heavy chain subclass and can therefore binds up to 60% of IgG antibodies from human serum sample.
The main goal of our work was the preparation and characterization of CIM Protein L disks. First, Protein L disks with different densities of Protein L on the support were prepared in order to define the dependance of the IgG capacity on the amount of the bound Protein L. Further on, the method of characterization of Protein L disk using IgG was developed. In the end, the stability of the developed CIM Protein L disks in different solutions was tested in order to define the operating and storage conditions.
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.