Posters

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.

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.

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.

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.

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 [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.

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.