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2023

The main objective of every downstream process (DSP) for AAV is to achieve high recovery while delivering the purest, most potent product possible. The capture step in AAV gene therapy is either affinity or cation exchange chromatography, which both concentrates the product and removes impurities. Following the capture, the eluate is generally further processed to enrich for full capsids and further purification. For this enrichment and polishing of full AAV capsids, CIMmultus QA, a monolith-based anion exchange chromatography, is widely used.

Since the polishing step relies on only small differences in charge of the AAV capsids, any process-induced heterogeneity or charge modulation of the capture eluate will diminish the separation efficiency and affect the step’s robustness. The affinity elution sample is reported to contain additional impurities,  which influences subsequent steps of the DSP. Processing time is critical to an efficient process since a faster process has an overall lower financial footprint.

A side-by-side comparison was performed using CIMmultus SO3-1mL (2um) column and commercially available affinity resin which binds several AAV serotypes. Both columns were evaluated for process and step recoveries, impurity reduction, product capacity and processing time. The results shown are based on two parallel experiments for each capture approach.

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Newcastle disease virus (NDV) from the Paramyxoviridae family is a single-stranded, negative-sense, enveloped RNA virus of avian origin. There are many commercial vaccines based on live or inactivated virions that are used worldwide to protect against its high morbidity and mortality rates among infected poultry, thus minimizing the economic loss. Due to strong host-range restriction, NDV is highly attenuated in humans and other primates, therefore it has been evaluated as a vector for several vaccines and is also promising as an oncolytic vector. Whether for vaccines or viral-vector therapies, NDV particles must meet certain criteria for yield, purity, and concentration. Having this in mind, the novel avian suspension cell line CCX.E10 is a significant advancement; the cell line is not a genetically modified organism (GMO), it comes in suspension culture or anchorage-dependent version and it grows in commercially available serum-free and animal-component–free media. When infected with NDV, CCX.E10 cells give high-titer harvests. With a combination of production on a CCX.E10 cell line from Nuvonis, which enables drug manufacturers to operate independently of SPF chicken-egg supply, and CIMmultus SO3 monolith technology, we developed a highly effective purification process for NDV.

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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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Adenovirus vector, a well-known gene therapy tool, vaccine vector and an oncolytic virus has recently regained attention during the global health crisis induced by Coronavirus. Their ability to induce a potent immune response is advantageous, and millions of doses have been administered worldwide with low rates of adverse effects observed [1]. Nonetheless, several serious thromboembolic events that were not correlated with mRNA-based vaccines have been described [1–5]. It has been indicated that the side effects could have been due to process-related impurities in the vaccines, such as host cell proteins [6] and DNA [5]. Regardless of the application, it is of the utmost importance to further improve the purity of the final product and reduce the possibility of potential side effects. In this work, we showcase a second-generation Adenovirus purification process based on CIMmultus® QA monolith chromatography (Figure 1). We additionally focus on bridging upstream and downstream using the newly developed PATfix® Adenovirus analytics, which allowed connection and enrichment of data obtained through commonly used methods such as ddPCR, total protein and infectivity assay.

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Quaternary amine (QA) modified monolithic columns are frequently used for purification of therapeutically relevant adeno-associated virus (AAV) vectors, used in gene therapy. Beside process- and sample-derived variability, chromatographic material variability can influence the efficiency and scalability of AAV downstream processing. This study presents the evaluation of highly reproducible (HR) QA-modified CIM monolithic columns, where the goal is to elute AAV  capsids in a very narrow conductivity range, regardless of the batch and size of the column used.  We have developed a chromatographic test method for proving the intra- and inter-batch homogeneity of the material through different column sizes. The method is based on separation of AAV2/8 capsids in ascending KCl gradient on CIMmultus QA 1 mL columns and specimen 0.2 mL units, taken from large-scale QA monoliths up to 8000 mL in size.

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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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Lentiviral vectors are efficient tools for transfer and stable integration of large gene inserts into the genomes of both dividing and non-dividing cells. Third generation of lentiviral vectors, which uses a fraction set of HIV genes, is replication incompetent and self-inactivating, offering a relatively safe tool for academic and industrial use, while offering a larger gene transfer capabilities compared to more commonly used AAV. As a result, several therapies using lentiviral vectors are already approved or in clinical trials, with primarily ex-vivo use. Limitations for more widespread use, as well as development of in-vivo therapies, however remain, with one of the causes being downstream purification of lentiviruses. Here we showcase a process development for CIM monolithic columns for use in downstream purification of lentiviruses. Several CIM monolithic chemistries have been tested to determine their performance for lentiviral purification, with three chemistries showing potential for further use. Of the three chemistries, two have been selected for further development. Several modifications of the two chemistries have been prepared and tested on CIMmic columns with promising initial results. The chemistries have been successfully upscaled to CIMmultus monoliths and the results from initial findings confirmed. Additional experiments are required to confirm the findings and perform optimization. The full downstream process will be developed once the most suitable chemistry is selected. During preparative, multi-angle light scatter (MALS) was used as indicator of viral presence, while analytics, used in the process development, were ddPCR and infectivity test.

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Several recombinant AAV (rAAV)-mediated therapies have been approved so far, and many more are in clinical trials, yet developing an efficient rAAV process platform remains a challenge. First obstacle arises from frequent practice of upstream (USP) and downstream (DSP) process being developed separately. USP optimization activities are usually focused on achieving high viral titer, which does not always translate into maximal purity and recovery in DSP. Second significant challenge is to bridge USP and DSP into a single bioprocess, due to lack of appropriate analytical methodology capable of producing reliable and unbiased results on both sides of the bridge. Furthermore, rAAV-based gene therapy vectors require the removal of process-specific combined with product-specific impurities, as they represent serious safety threats. Here we showcase the example of AAV8 USP optimization directed towards good performance in DSP. AAV8 is produced in HEK suspension cells via plasmid transfection. Based on the screening results, the best performing set of conditions was successfully scaled up to a 5L stirred-tank bioreactor. Harvested material was then evaluated in DSP, comprised of pre-capture, and two ion-exchange chromatographic steps on CIM monolithic columns. As bridging analytics of choice, we relied on PATfix platform, (d)dPCR and Comassie Bradford protein assay measurements for thorough assessment of relevant parameters such as titer, percentage of full AAV8 capsids and total protein concentration.

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In every adeno associated virus (AAV) downstream process the key steps are the enrichment of full capsids and the in-process empty/full analytics. A common approach for enrichment of full capsids is liquid chromatography using ion exchange or multimodal chemistries, which based on particles’ surface charge differences, enables separation of full (F) capsids and product related impurities. Removal of impurities is an important step as they may pose a serious health risk, as well as deteriorate the economics of the production process. A PATfix® triple detector set in combination with an CIMac™ AAVEmpty/Full analytical column by Sartorius BIA Separations was used to determine E/F ratio. The column is a strong anion exchanger which exploits capsid charge differences for separation when employing an ascending salt gradient. Usually, NaCl with the addition of 2 mM magnesium is used, as the presence of magnesium has been shown to improve the peak shape and the removal of empty capsids. According to the Hofmeister series salts have different impact on non-covalent interactions among capsid themselves and between columns and capsids. For example, antichaotrope salts were shown to improve the empty/full capsid ratio, possibly by inducing the hydrophobic interactions between them. Quaternary alkyl ammonium salts (QAAS) are known to have antichaotropic properties and they differ in the substituents. Wang et al. reported that QAASs produced a better empty/full separation than NaCl. This poster presents a screening of different eluents for empty/full analytics. Beside NaCl as a reference salt, two QAAS were chosen: tetramethylammonium chloride- TMAC and a possible nonhazardous alternative choline chloride, which is bifunctional and contains both a quaternary ammonium and hydroxyl groups. The influence of magnesium on E/F separation was also tested.

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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.

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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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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.

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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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Monoliths are commonly used for separation of empty and full AAV capsids on analytical and preparative scale. PrimaS approach for AAV separation employs an ascending pH gradient, which can be sensitive towards small changes in chromatographic parameters. In present work 200 µL testing units extracted from large CIMmultus PrimaS monolithic columns, were employed for the adjustment of critical chromatographic parameters.

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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.

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Standard 96-well design offers a great advantage for screening many samples or conditions and supports process automatization. Our approach was multi-parallel screening of different mobile phases for rAAV capture step using CIM® SO3 0.05 mL Monolithic 96-well Plates. Buffers of different pH, sodium chloride concentrations and use of Poloxamer 188 were screened to purify AAV2/9 clarified lysate obtained from Sf9 cells. Sample was pretreated by tangential flow filtration (TFF) coupled with nuclease treatment – Kryptonase. It was shown that the optimal conditions were buffers of pH 3.5, 500 mM NaCl, with addition of Poloxamer 188. Verification of results with selected buffer resulted in high capacity (1.44E14 capsids/mL SO3), great recovery (87.7 %) and excellent protein and DNA reduction (99.98 and 99.25 %).

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In each adeno associated virus (AAV) downstream process one of the key steps is enrichment of full capsids. This could be achieved by density gradient ultracentrifugation however a main drawback is its scalability. A more common approach is liquid chromatography using ion exchange chemistries, which based on particles’ charge differences, enables separation of full (F) capsids and product related impurities including non functioning AAV capsids (empty, partially filled, misfolded and wrongly packaged genome or other DNA containing subspecies).

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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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