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2024

Lipid nanoparticles (are leading non viral carriers for therapeutics, offering versatility in encapsulating diverse payloads Their manufacturing superiority over viral systems allows for modularity, speed, and scalability However, this modularity poses challenges in purification and characterization due to sample uniqueness LNPs require downstream processing for in vivo application and adherence to critical quality attributes ( Analytical methods for those currently predominantly require undesirable particle disassembly beforehand.

Monolithic columns offer ideal chromatography for LNPs due to laminar flow, minimizing shear forces, and surface modification enabling selective options Here is presented the purification method for LNPs on monolithic columns utilizing the PATfix® analytical chromatographic system, efficiently separating LNPs from free cargo.

An analytical scale two dimensional chromatographic tool was developed It delivers comprehensive characterization of encapsulation efficiency, nucleic acid content, degradation, and separation of co encapsulated cargos, without any sample pre treatment Highly tunable and automatable, this method maximizes efficiency and facilitates precise separation of LNP populations.

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2023

Immobilized enzyme reactors (IMERs) stand as innovative biotechnological constructs, seamlessly merging the catalytic proficiency of enzymes with the advantages of solid support matrices. Immobilized enzymes offer notable benefits such as improved stability, the potential to operate within a continuous system over extended durations, reusability of the enzyme, as well as reduced production costs and product purification steps. The aim of this study was to prepare a functional IMER on monolithic support for efficient pDNA linearization, that could be used in in vitro transcription reaction for messenger ribonucleic acid (mRNA) production.

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New development in the modern biotechnology increased the need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA), but their chromatographic purification is often challenging due to low process yields and column clogging. There are 
indirect proofs that open circular (OC) pDNA isoform is the main troublemaker due to its physical entrapment within the narrow channels of chromatographic media. Increasing the channel size of chromatographic support should decrease the negative 
impact and improve the chromatographic performance. The aim of the study was to use novel Convective Interaction Media® (CIM®) monolith chromatographic columns with large, 6 µm channels, for analytical and preparative separation of pDNA. The effect of supercoiled (SC), OC and linear (LIN) pDNA isoforms on chromatographic performance was thoroughly evaluated.

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2022

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.

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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. 

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2021

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.

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2018

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.

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2017

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.

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2016

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.

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2014

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.

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2012

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.

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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.

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2010

Application of plasmid DNA for gene therapy and vaccination has gained huge interest in last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Major influence on achieving regulatory demands in pDNA production has downstream processing and in order to get optimal purity different purification techniques have to be included. It was demonstrated that methacrylate monoliths can be used for efficient purification process of plasmid DNA. High dynamic binding capacities and high flow rates of methacrylate monolith enabled excelent purity and productivity.

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2009

Application of plasmid DNA for gene therapy and vaccination has gained substantial interest in the last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Downstream processing has major influence on achieving regulatory demands in pDNA production and in order to get optimal purity different purification techniques have to be applied. It was demonstrated that methacrylate monoliths can be used for efficient purification process of plasmid DNA. High dynamic binding capacities and high flow rates of methacrylate monolith enable excellent purity and productivity.

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2008

Anion-exchange chromatography is fundamental in downstream processing of plasmids both as a process and analytical technique. CIM anion-exchange monolithic columns have already been successfully used for the industrial scale purification of pharmaceutical grade small plasmid DNA [1].

In this work we report about the use of the newly developed monolithic analytical column intended for plasmid DNA determination in terms of its analytical performance. Higher degree of sensitivity, precision and accuracy is necessary in order to determine the quality of clinical grade DNA intended for therapeutic use. Plasmids purified from Escherichia coli fermentation exist predominantly in the supercoiled form (SC) the other two topoisomers present in the final product are mostly the open circular (OC) and linear forms [2]. Different chromatographic conditions were tested and the separation was optimized in terms of buffer and pH selection as well as in terms of gradient slope and column length. The results were compared to the results obtained with established analytical methods.

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2006

Gene therapy has already shown some great results in treatment and cure of some monogene diseases, such as diabetes. While the use of genetically modified viruses raises safety concerns, synthetic formulations of genes inserted in plasmids are regarded as safer. At present, most clinical trials involve plasmids smaller than 10 kb. However, the concern that regulation of the functioning of the gene is ensured together with the expectation of the progression of gene therapy to multigene disfunctions, like cancer or complex nevrodegenerative disfunctions (Alzheimer disease), will require the production of larger plasmids [1].

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2005

Plasmids are excellent genetic vectors and have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years, plasmids are intensively investigated for gene therapy purposes and genetic vaccination. In this case, plasmid DNA (pDNA) of high purity is required. To follow such demands, several chromatographic steps are commonly needed. In the case of buffer compatibility, columns can be connected in-line to overcome time consuming and yield lowering multiple chromatographic steps. Since each of the unit operations contributes to the dispersion, the resolution is further decreased by each chromatographic step. This drawback might be surmounted by combining several chromatography steps into a single chromatography column. This approach is known as multidimensional or conjoint liquid chromatography (CLC).

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2004

By using a combination of two CIM® tube monolithic columns, OH and DEAE chemistry, we were able to successfully purify plasmid DNA from bacterial culture without using RNase. Purified plasmid DNA is very pure, since common contaminants, such as proteins, genomic DNA, endotoxins and RNA were under the detection limit. The scale up units produced according to cGMP standard are already used for the purification of plasmid DNA for gene therapy purposes on industrial scale.

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2003

Gene therapy which is becoming more and more important in human health care requires the purification of high molecular mass compounds, so called nanoparticles (e. g. viruses and plasmids). The method of choice to ensure proper purity would be chromatography.

Most of the chromatographic supports available on the market at the moment can not follow the requests for such work due to low binding capacity for large molecules, limitation with regards to the time of the separation process and requests for CIP (cleaning in place) and SIP (sanitation in place).

Monolithic supports represent a new generation of chromatographic supports. In contrast to conventional particle supports, where the void volume between individual porous particles is unavoidable, these supports consist of a single monolith highly interconnected with larger and smaller open flow-through channels. Due to the structure, molecules to be separated are transported to the active sites on the stationary phase by convection, resulting in very short separation times. This is especially true for large molecules.

In this work we will present the use of monolithic supports for the separation of different nanoparticles on analytical and preparative scales. It will be shown that monolithic supports can overcome the limitations of particle-based supports for the analytics and isolation of big molecules and represent a major step towards the safe and efficient purification or production of nanoparticles.

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Plasmids are episomes that have been recognized in few eukaryotic and most prokaryotic species. Some plasmids are excellent genetic vectors and they have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years plasmids were intensively used for gene therapy purposes (1). Most often purification starts with the cells harvest followed by alkaline lysis step in which ribonuclease A (RNase) is typically used. After that, plasmid DNA can be precipitated and used directly or can be further purified by different methods (2). Currently, several chromatographic methods, such as ion-exchange, size exclusion, affinity, and hydrophobic chromatography, have been demonstrated in plasmid purification (3). Until now a limited number of small scale purification methods without use of RNase were published. Convective Interaction Media CIM® is a monolithic chromatographic support for which has been shown that is very efficient for the separation of large molecules, such as proteins, DNA and viruses (4).

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