Posters

mRNA has been at the forefront of both scientific and general public interests from the start of the COVID-19 pandemic. However, there are still limited options available for rapid characterization of mRNA containing samples. For precise characterization of an mRNA sample, first the presence and concentration of mRNA molecules in the sample needs to be identified. In the second step, any contaminants in the sample coming from the IVT reaction need to be identified and quantified. All major components of the IVT reaction; nucleotides, capping reagent, enzymes and DNA template may be present in the mRNA sample. In addition, impurities such as shorter, incomplete RNA fragments, and in particular, dsRNA may also be present. Contaminants may also come from the mRNA in vitro instability, caused by spontaneous hydrolyzation of the mRNA backbone. These issues can be mitigated using appropriate analytical tools throughout the mRNA production and purification steps.

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.

Adenovirus is well-know gene therapy tool that gained attention as a promising vaccine delivery vehicle, specially during Covid-19 pandemics, where it was used to deliver sequence for protein S (S). Multiple serotypes have been tested in clinical trials for various applications, the most common one being human adenovirus serotype 5 (Ad5). With this in mind, we chose Ad5-S construct with GFP tag as a model vector to develop upstream process (USP) and supporting analytical tools.

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. 

Recombinant adeno-associated viral (rAAV) vectors are the leading gene delivery tool for treatment of a variety of diseases. While several rAAV mediated therapies have been approved so far, and many more are in clinical trials, rAAV production still faces many challenges. Key goal of rAAV upstream process development is achievieng high viral titer together with a sufficient percentage of full capsids. Moreover, analysis of complex upstream harvest samples can be challenging. Classical analytical methods such as ddPCR/ELISA offer limited information due to differences in sample preparation and basic principles for detecting empty and full capsids. Method is also time consuming and therefore less useful for following rAAV production process in real time. To overcome these limitations, we developed a PATfix Valve Switch analytical method that is based on ion exchange biochromatography and can be successfully applied for analysis of empty/full ratios in crude upstream samples.

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 system using PATfix size exclusion (SEC) analytical method.

The IVT reaction is one of the most expensive steps in mRNA production process and its optimization to reach high mRNA yield is of key importance Standard mRNA quantification techniques like absorbance and fluorescence based assays are time consuming and cannot be performed at line as the IVT reaction progresses In addition, other reaction components like nucleotides and pDNA interfere in the analytical results and reduce the method’s accuracy A new approach shown here uses CIMac PrimaS™ analytical HPLC column to separate and quantify several key IVT components with a very short run time, enabling fast “at line” tracking

Density gradient ultracentrifugation (DGUC) is a well established tool for Empty/ Full AAV capsid separation based on density differences between AVV sub-populations. However DGUC practice is laborious and lacks any detection options, therefore fractions must be collected manually and analyzed later. Both of these shortcomings can be addressed by coupling post DGUC workflow to PATfix analytical system. BIA Separations PATfix platform provides sufficient tools for liquid extraction and fractionation as well as a comprehensive detector suite for precise fraction characterization. Baseline separation of capsid species was achieved in a density gradient of CsCl, producing a centrifugram that reveals information traditional DGUC and anion exchange chromatography cannot.

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.

PATfix with convective chromatography media (e.g.monolith) offers a rapid analytical method to characterise complex mixtures. Transcription reaction used for production of mRNA represents such a mixture, with components varying in size, chemical and physical properties. A new analytical approach (PATfix) presented here uses CIMac PrimaS to separate IVT components such as triphosphate-nucleotides (NTPs), enzymes, DNA template and RNA in a very short gradient.

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).

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.