Posters

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Monolith chromatography media coupled with metal affinity ligands proved superior to the conventional particle-based matrix as a plasmid DNA (pDNA) purification platform. By harnessing the differential affinity of pDNA, RNA. Host cell proteins and endotoxin to copper ions in the solution a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl2-induced precipitation. RNA and remaining endotoxin were subsequently processed by copper immobilized metal affinity column employing either monolith or particle-based matrix where both RNA and endotoxin were removed below detection limit with almost complete recovery of pDNA in the monolith was found to have several advantages in terms of handling feedstocks crowded with RNA in a concentration-independent manner and exhibiting flowrate-independent dynamic binding capacity for RNA. This enabled monolith-based process to be conducted at high feed concentration and flow rate. Resulting in pDNA vaccine purification at a high yield and purity and the process conditions investigated, the use of monolith column gave at least three fold higher productivity for recovery of purified pDNA as compared to the particle- based column, demonstrating its potential as a more rapid and economical platform for pDNA vaccine purification.

The present study describes a new methodology to quantify and monitor the quality of supercoiled (sc) plasmid DHA (pDLIA), using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows distinguishing the plasmid isoforms by a NaCl stepwise gradient. The selectivity, Linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a sc plasmid concentration up to 200 µg/mL. The main advance achieved by using this monolithic method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, in a 4 minutes experiment. This work also intends to evaluate the possibility to assess the sc pDNA present in more complex samples, allowing the control of the samples recovered from different bioprocess steps.