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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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Extracellular vesicles (EVs) are a diverse group of particles secreted by all living cells. Numerous different therapeutic applications of both native and engineered EVs are currently in different stages of clinical development. Nevertheless, considerable challenges are still present in the manufacturing, purification and analytics of EVs. Many factors can influence the final product, therefore an all-inclusive approach to development of the bioprocess is needed. Cell culture parameters and production platform selection might alter the number and composition of EVs. Furthermore, raw materials used in upstream production, such as media and supplements, can greatly impact the chromatographic purification. In this study, we evaluated EV production in different HEK293-derived cell lines. Separation on a strong anion exchange column CIMmultus®-EV was used to assess the abundance of different EV populations. Multi-detector PATfix® SEC analytics coupled with antibody labeling was then used to analyze chromatographic fractions. Furthermore, the analytical methods and performance in downstream processing were applied in the optimization of the upstream process.

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Cells release extracellular vesicles (EVs) of different sizes and intracellular origin. Due to their heterogenicity, the isolation of the target EV population from a mixture of supernatant-derived particles can be challenging. Anion exchange chromatography (AEX) exploits the negative charge on EV surface molecules for binding to the positively charged solid phase. CIMmultus® EV, an AEX chromatography monolith column, can separate EVs in subpopulations based on charge and offers insight into the heterogenicity of particles. Besides the availability of preparative tools for separation, combining multiple orthogonal and complementary characterization tools is crucial for defining the EV product of interest. In this work, we used a multiple-detector PATfix® system for the analysis of CIMmultus EV-fractionated samples. Samples were analyzed for the presence of EV-related tetraspanins using the fluorescence detector. PATfix MALS 3609 detector was used for the analysis of particle-containing samples and calculation of particle sizes.

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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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Endotoxins are robust and persistent impurity, which are native to majority of phage substrates. Two anion exchangers, CIMmultus PrimaS and H-Bond, were tested for their capacity for endotoxin removal in comparison to well known strong anion exchanger, CIMmultus QA. 

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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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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.

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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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2020

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.

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2019

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.

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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.

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One of the handicaps of working with bacteriophages is the long duration required to perform plaque assays. Plaque assays also impose questions about accuracy and precision relative to the scale and experience of the persons performing and interpreting them. This poster presents a pair of high precision, high accuracy chromatography-based assays that permit determination of phage concentration in less than 1 hour. Sensitivity of UV absorbance is poor because of the low concentration of phages. However, phage sensitivity is strongly amplified by monitoring the chromatogram with either fluorescence or MALS. Fluorescence works by measuring the fluorescence emission from tryptophan residues of the phage proteins. MALS works by passing a laser beam through the sample and reading the scatter produced when it encounters a particle. Larger species generate more scatter.

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Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants. The most common technique for purification is ultracentrifugation using cesium chloride gradients. This technique is elaborate, cumbersome, expensive and difficult to scale-up.
Alternative techniques for purification are usually time consuming and affect phage recovery and/or viability. In this study we present efficient two-step chromatographic purification method with binding phages to a stationary phase - Convective Interaction Media (CIM®) monoliths. The aim of the study was to develop robust, fast and effective virus purification platform that can be used for several types of bacteriophages for any application. In this work bacterial lysate with bacteriophage T4 (host E.Coli) was used.

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2018

Immunoaffinity columns using antibodies as ligands against mammalian proteins could be used for different applications in protein expression control and, if a standard available, for direct protein quantification in complex sample solutions. Additionally, these columns are ideal for polishing step of recombinant proteins, such as mammalian receptor Fc fusion proteins. Most importantly, such columns could extract a significant amount of a single membrane protein from native source, suitable for downstream analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against RAE-1 GPI anchored glycoprotein were developed (CIMmic HDZ - @RAE-1 column) as a part of Glycomet project with the main goal to analyze the antigen glycoprofile.

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Hydrazide-activated (HDZ) columns were proven to be a product of choice for making the most effective immunoaffinity columns. They take advantage of a special hydrazide linkage that binds antibodies through the carbohydrate residues on their Fc regions. This leaves the antigen-binding domains fully accessible to enable the most effective capture of desired target (Figure bellow).
CIMac™ HDZ monoliths make HDZ-immobilized antibody columns even more effective. Because of their large channel size and the efficiency of convective mass transport, they eliminate the long loading residence times that are required for affinity chromatography on porous particle columns. Flow rates of 5–10 column volumes per minute allow complete purifications in a few minutes, even when the source material contains a low concentration of antigen. The same performance is achieved whether a small peptide or a large bio-assemblage like a virus particle or extracellular vesicle is isolated. The combination of HDZ monoliths and the immobilization protocol offers a strong tool for fast antigen isolation from complex biological sample (plasma, lysate, etc.) and consequently sensitive antigen quantification. An example of CIMac™ HDZ application is a purification of fibrinogen from human plasma.

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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

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.

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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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