Posters

The increasing demand for messenger RNA (mRNA) as a therapeutic product requires larger production scales and more efficient extraction techniques. In this poster, fast and efficient way to purify poly-adenylated mRNA using affinity chromatography on CIMmultus™ Oligo dT column is presented.

The poly-adenylated tail of mRNA interacts with covalently bound oligo dT ligands in high-salt loading conditions, where electrostatic repulsion between negatively charged backbones of both, mRNA and oligo dT, are reduced and H-bonding in T-A base pair is emphasized. High salt concentration additionally screens out attractive electrostatic interactions between mRNA and other components in the process sample, thus facilitating aggregate reduction in purified product.

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 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.

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).

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 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.

Production of high value biological therapeutics usually involves complex manufacturing processes with high process variability. Additionally, development of robust and reliable bioprocesses can be challenging. PAT aims to enhance bioprocess understanding and implies a holistic approach to ensure that quality is built into products by design. Efficient PAT therefore calls for fast and robust analytical techniques which enables to asses high quality information about critical quality attributes and key performance indicators as parallel as possible to the manufacturing process. PATfix™ is unique analytical system for routine gradient separations that enables every analytical task. Equipped with bio-inert ceramic pump heads is deliberately tailored to meet the demands of analytical applications covering wide range of biomolecules. Highly sensitive and fast multi-wavelength detector enables to detect component peaks even in very fast gradients.

Productivity of the downstream bioprocessing depends among others on the efficiency of chromatographic step. One of the crucial chromatographic parameters is dynamic binding capacity (DBC) for certain biomolecule. DBC could be tailored with changing the surface area of convective pores by tailoring the surface of pre-polymerized monoliths using graft or block polymerization of polymer brushes. Grafted CIM monoliths have already been prepared via Radical Polymerization (RP) and successfully characterized (1).

Recently, the implementation and optimization of Controlled Radical Polymerization (CRP) for grafting of large pore monoliths (average diameter 6 μm ) resulted in polymethacrylate-based ionic exchanger with at least 5 times higher DBC compared to non-grafted 6 μm monoliths, while preserving high permeability. The main goal of our study was to chromatographically characterize novel grafted ion-exchanging monoliths (CIM gDEAE and CIM gSO3) to see whether novel columns still retain flow independent chromatographic properties of non-grafted monoliths.

To ensure the desired chromatographic characteristics of the CIM® monolithic column at large scales, monolith microstructure morphology, pore size distribution, porosity and surface ligand density should be uniform. To demonstrate the uniformity of large chromatographic monoliths we have developed new testing procedures. By fabricating smaller columns (disks) from different random  positions of larger monolith, non-cGMP compliant chromatographic testing can be applied on the same polymerization batch without affecting the cGMP compliance of large-scale chromatographic monolith. Each individual disk was thoroughly tested and the results were compared to the properties of the large monolith.

There are many cases, where a single protein needs to be purified from a complex sample. Such proteins manifest themselves as impurities, which can affect further analysis, either by causing specific equipment malfunction or lower yield in the products. In other cases the specific protein is our molecule of interest, for example in glycomics analysis. In both cases high specificity for proteins, reproducibility and reliability is necessary. We have developed a model immunoaffinity column and 96-well plate based on an anti-fibrinogen monoclonal antibody, covalently immobilized onto CIMac™ analytical chromatographic monolith.

There are many cases, where a single protein needs to be purified from a complex sample. Such proteins manifest themselves as impurities, which can affect further analysis, either by causing specific equipment malfunction or lower yield in the products. In other cases the specific protein is our molecule of interest, for example in glycomics analysis. In both cases high specificity for proteins, reproducibility and reliability is necessary. We have developed a model immunoaffinity column and 96-well plate based on an anti-fibrinogen monoclonal antibody, covalently immobilized onto CIMac™ HDZ analytical chromatographic monolith.

Methacrylate monoliths (CIM® monolithic columns) allow for very fast and efficient separations and exhibit very high binding capacities for extremely large bio-particles due to their large inner channel diameters and enhanced mass transfer characteristics.
Additionally, the ability to manufacture polymer monolithic materials ranging from analytical to large scale preparative/industrial columns has tremendous advantages. By ensuring the chromatographic properties are consistent over the whole size range, one can easily design and optimize a purification method on laboratory scale and transfer it to a production line with minimal to no additional modifications.

Until now the largest monolithic column had a volume of 8 L, which was large enough to serve the biopharmaceutics' market's needs. Now however, the capacity of that column is already at its upper limit.

By successfully employing the knowledge and experience from almost two decades of monolith production we have managed to overcome the size limitations and polymerize the largest convective chromatographic support made from one piece of material, a 40 L monolithic column.

Immunoaffinity columns using antibodies as ligands against mammalian membrane proteins could be used for different applications in protein expresion control and, if a standard available, for concentration determination. Additionally these columns are ideal for polishing step of Fc fusion proteins of mammalian receptors.

Most importantly such columns could extract a significant amount of a pure membrane mammalian protein suitable for structural analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against MULT-1 transmembrane and RAE-1 GPI anchored glycoproteins were developed as a part of Glycomet project with the main goal to analyze the antigen glycan parts.

Two different  preactivated support were used:  hydrazide (HDZ) and carboxy imidazole (CDI).

Surface hydrophobicity/hydrophilicity of chromatographic stationary phases is one of the important characteristics that influence the chromatographic column performance. On the one hand, the surface should be highly hydrophilic to avoid nonspecific adsorption of sample molecules; on the other hand, the hydrophobic surface is crutial to e.g. separate the molecule isoforms.Therefore, fast and easy characterization method to evaluate the surface „hydrophobic/hydrophilic character" could be valuable.

First stage in the development of this method and the objective of this study was to evaluate the hydrophobicity of test set of 1 mL CIM columns with different ligand chemistries and densities. This was achieved by separation of protein mixture under hydrophobic interaction chromatography (HIC) conditions. Proteins were used since monoliths are used mainly in downstream of large biomolecules.

Moreover, since poor recovery under HIC conditions was observed on some columns, the research was additionally expanded with reversed phase chromatography (RPC) to obtain extra information about even more hydrophobic surface properties of monolithic columns. Therefore, after HIC step the RPC step followed and additional elution of proteins was achieved.

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.

Phosphoproteomics is a branch of proteomics that focuses on deriving a comprehensive view of the extent & dynamics of protein phosphorylation by way of identifying & characterizing proteins that contain a phosphate group as a posttranslational modification. One of the approaches for specific enrichment of phosphopeptides from complex samples is metal oxide affinity chromatography (MOAC), where the specific adsorption results from bridging bidentate bindings formed between the phosphate anions and the surface of a metal oxide, such as TiO2, ZrO2, Fe2O3, and Al2O3. In presented study, a rutile TiO2 nanoparticles were bound to a previously polymerised CIM hydroxy monoliths.

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.

Challenges in monitoring the quality of vaccine production
• Process Analytical Technology (PAT) ensures process reproducibility in bioprocessing
• A mechanism to design, analyze and control pharmaceutical manufacturing processes through the measurement of critical process parameters (CPP) which affect product quality attributes (CQA)
• Initiated by the FDA as part of the 21st Century GMP initiative in 2001 with the goal of increasing productivity
• Application of PAT in vaccine development and manufacturing is challenging due to the sample complexity and batch-to-batch variability.
• During the development of an up- and/or down-stream process of the target biomolecule, a fast, accurate and reliable analytical method is requried for determining the quantity and purity of the product intended for human use

Solution: Convective Interaction Media Monoliths
• Monoliths are chromatography media cast as a single block, inserted into a housing
• Highly inter-connected network of channels (1-2 μm) containing functionalised binding sites for large biomolecules (viruses, VLPs, pDNA, antibodies)
• Performance unaffected by increasing the flow rate or molecular size

There are two different designs of chromatographic columns concerning the flow profile. Most of today's HPLC columns belong to the group of so-called axial mode operating columns, while the radial ones with a radial flow pattern are more rare. Which type performs better depends on the particular case but it seems that the radial operating columns are attracting interest since they exhibit some beneficial features. One of the main problems of radial operating chromatographic columns is the changing of a mobile phase linear velocity over the chromatographic bed. Because of that, matrix efficiency for porous particulate supports varies by its position within the bed, and overall performance is more difficult to predict.

This problem is not present when the monolithic supports are used, since it was demonstrated that their chromatographic properties are flow unaffected even at the extreme linear velocities. This was confirmed also for the radial operating mode.

The monolith and radial flow housing were designed for extremely high flow rates, up to 70 CV/min, which is the range of the flow rates applied on membranes. This was achieved by proper monolith dimensions with the height of 55 mm, inner diameter of 6.0 mm and thickness of only 4.5 mm.

In the last few years pharmacology has made a big step towards the new type of drugs, called biological drugs. Popularity and market for biological drugs grew exponentially, so did the need for fast and inexpensive purification. Classic liquid chromatography columns were unable to separate biological compounds in industrial quantities, therefore the scientists were looking for alternatives. One of them are monolithic materials. Monolithic materials, especially methacrylate monoliths, are becoming more and more popular in separation processes due to their fast separations, low pressure drop and mechanical stability.

In the context of preparing new columns and improving existing ones, we need to know every single chemical as well as mechanical property of our monolithic material. Here we present some key data and interesting correlations between mechanical and structural properties of GMA-co-EDMA porous monolith. In the first paragraph we compare nonmodified and DEAE modified monoliths with different average pore size and porosity, regarding to their compression and tension properties. The second paragraph deals with the impact of these parameters on the permeability of the column during separation.

Commonly, epoxide-based monoliths used as porous supports in affinity chromatography are synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) by free radical polymerization.

We prepared an epoxide-based monolith by self polymerization of polyglycidyl ethers where the epoxy groups serve as functional groups for the polymerization reaction as well as for the immobilization of the ligand.