Posters

Sample displacement chromatography (SDC) is a chromatographic technique that utilizes differences in relative binding affinities of components in a sample mixture under chromatographic conditions. Here, we use SDC approach with CIM® C4 HLD monoliths under hydrophobic interaction chromatography (HIC) conditions to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (SC) isoform acts as a displacer of open circular (OC) or linear isoform. High purity of SC isoform is beneficial for use of plasmids as vaccines, transfecting agents for production of gene therapy viral vectors, or as starting material for linearization prior to IVT reaction in production of mRNA vaccines.

Endotoxins are robust and persistent impurity, which are native to majority of phage substrates. Two anion exchangers, CIMmultus PrimaS and H-Bond, were tested for their capacity for endotoxin removal in comparison to well known strong anion exchanger, CIMmultus QA. 

Agarose gel electrophoresis (AGE) analysis is an important method for monitoring of plasmid DNA (pDNA) quality, with ability to separate pDNA isoforms (sc, oc, lin, multimer). Plasmid linearization can be monitored for purposes of producing starting material for mRNA production. Electrophoretic conditions and, more importantly, matrix used for sample dilution before gel loading can affect analytical results. We have observed that purified linear pDNA shows an additional band in AGE analysis of the sample in water medium, which can lead to misinterpretation of results. 

Optimizing processing steps in sc pDNA isolation is critical for obtaining good process yields as well as high product purity. PATfix platform with convective chromatography media (e.g. monolith) offers a rapid analytical method to characterize complex biomolecular mixtures and gives immediate feedback during process development. E coli lysis represents such a challenging step, where multiple critical quality attributes need to be identified and critical processing parameters optimized. This approach leads to better yields and product purity, allowing for simplified downstream steps. A new PATfix analytical platform presented here uses CIMac pDNA column, to separate and characterize plasmid from impurities, allowing for easy optimization of key parameters such as RNA removal.

One of the handicaps of working with bacteriophages is the long duration required to perform plaque assays. Plaque assays also impose questions about accuracy and precision relative to the scale and experience of the persons performing and interpreting them. This poster presents a pair of high precision, high accuracy chromatography-based assays that permit determination of phage concentration in less than 1 hour. Sensitivity of UV absorbance is poor because of the low concentration of phages. However, phage sensitivity is strongly amplified by monitoring the chromatogram with either fluorescence or MALS. Fluorescence works by measuring the fluorescence emission from tryptophan residues of the phage proteins. MALS works by passing a laser beam through the sample and reading the scatter produced when it encounters a particle. Larger species generate more scatter.

Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants. The most common technique for purification is ultracentrifugation using cesium chloride gradients. This technique is elaborate, cumbersome, expensive and difficult to scale-up.
Alternative techniques for purification are usually time consuming and affect phage recovery and/or viability. In this study we present efficient two-step chromatographic purification method with binding phages to a stationary phase - Convective Interaction Media (CIM®) monoliths. The aim of the study was to develop robust, fast and effective virus purification platform that can be used for several types of bacteriophages for any application. In this work bacterial lysate with bacteriophage T4 (host E.Coli) was used.

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.

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.

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.

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.

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.

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.

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 [1].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.