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2022

Robust and precise chromatographic analytical methods are key for efficient development of mRNA production process. Three different analytical methods, which utilize three different column chemistries, are embedded in a ready-to-use PATfix™ mRNA analytical platform to support mRNA process development and product quantification and characterization.

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First chromatographic step in industrial platform using monoliths is a capture step, where rAAV is selectively bound to strong cation exchanger (SO3) in acidic pH conditions (1,2). In this step majority of host cell contaminants are removed and rAAV is strongly bound to the matrix. Productivity of the step is therefore strongly influenced by the dynamic binding capacity (DBC). DBC is dependent on the sample preparation prior to chromatography and the availability of the binding sites at the chromatographic matrix.

Due to increasing demand for rAAV quantity and quality for clinical manufacturing, large volumes of upstream produced material are being purified on industrial scale. At this point, rAAV capture is a first chromatographic step to be optimized for optimal selection of the chromatographic parameters. Doing this, SO3 monolithic columns can be used with their full potential. To reduce the development time using multi-factor screening and increase comparability during downstream process development, CIM® SO3 monolithic 96-well plates were introduced to Sartorius BIA Separations portfolio. Obtained results on small scale can be applied to the CIMmultus™ line, which is scalable to large industrial volume.

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Buffer conditions (salt, additives) influence mRNA binding on Oligo dT. Three contributing factors were identified and tested: NaCl, MgCl2 and Gu-HCl, the latter leading to a capacity of >6 mg/mL.

Affinity-based chromatographic isolation of mRNA is robust and simple, lending itself as a useful industrial platform. mRNA constructs contain a 3’ polyA tail to increase stability in vivo, thereby affording the possibility of affinity purification using oligo-deoxythymidinic acid (Oligo dT) probes covalently coupled to a solid support. Poly-adenylated mRNA forms a stable hybrid with Oligo dT under high-salt conditions which is destabilized when the salt is removed, allowing mRNA to be released.

Due to an increasing productivity of IVT reaction, finding conditions that increase binding capacity of Oligo dT has been an intense focus of development. Multi-parallel approaches, such as screening in multi-well plate format, can significantly cut the development time by screening multiple conditions at once. 96-well plates can then be scaled-up to preparative scale, such as CIMmultus™ product line operated by chromatographic skids.

Continue to products for screening

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2021

Optimized analytical methods are key components of a well-functioning analytical system, while method development usually comes with a time-consuming learning curve and optimization.

PATfix pDNA analytics platform, designed for in-process control of linear pDNA production, enables monitoring of pDNA linearization progression, as shown in Figure 1. Fully optimized and validated analytical methods, as well as guidelines for buffer and sample preparation come as part of the PATfix system, allowing users to focus on their specific application. In addition, the PATfix pDNA analytical package includes a pDNA calibration standard, which enables accurate quantification of the pDNA species of interest.

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2019

A purification of synthetic oligonucleotides by using CIM™ monolith was evaluated. In this case study, the CIM™ anion exchange column had the capability to resolve oligonucleotides with small difference in comparative chain length.

A crude reaction mixture of synthetic oligonucleotide was loaded onto the CIM™ anion exchange column. Sample elution was achieved by salt concentration gradient. In comparison with conventional media, CIM™ monolith indicated higher resolution for major impurities.

Advantages of the characteristic properties of the CIM™ monolith were evaluated based on the high throughput purification of oligonucleotides under the identified gradient separation conditions. Over 99 % HPLC purity for the target oligoDNA was achieved by one-step purification from the crude reaction mixture.

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Pre-activated CIMmic™ monolithic columns are cost efficient tools for screening of immobilisation conditions and small scale proof-of-concept testing of custom affinity columns and enzymatic reactors. Each column is assembled from a dedicated housing and discs containing a chromatography medium. With a bed volume of 100 μL, sample requirements are minimal, while inserting multiple discs in the housing adapts the column volume to application requirements. Different surface modifications of the discs enable immobilisation of a wide variety of ligands.

The increasing demand for messenger RNA (mRNA) as a therapeutic product requires larger production scales, and in turn more efficient extraction techniques. One of the most convenient techniques for its extraction is the use of oligo deoxythymidine (dT) coupled to a solid support [1]. Oligo dT hybridises to the poly-adenylated tail which is present on most eukaryotic mRNAs, or synthetised onto the molecule during IVT, while other contaminant impurities (proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes) lack the poly-A moiety and do not adhere to the solid support.

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2018

The application describes separation of Ni species by assembling four weak CIM DEAE anion-exchange disks into a monolithic column. The concentrations of the Ni species eluted from the column were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID)-ICP-MS. The Ni binding ligands eluted under the chromatographic peaks were identified off-line by tandem electro spray mass spectrometry (ESI-MS-MS), scanning for negative ions.
The mild chromatographic conditions of the CIM DEAE disks preserved chemical species and enabled separation of negatively charged Ni complexes.4 NH4NO3 was chosen as eluent since it enabled separation of Ni species and is compatible with ICP-MS and mass spectrometry detectors.

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2017

Human coronavirus OC43 (HCoV-OC43) is a frequent cause of respiratory tract illness, ranging from common cold to severe disease. The research on coronaviruses and medical application of coronaviral vectors/vaccines requires a quality material of high purity. Unfortunately, virus preparations are highly contaminated with cell debris and purification requires laborious, cost-ineffective procedures.
Here, we report a simple and efficient method for coronavirus concentration and purification by the example of HCoV-OC43. To achieve this, virus chromatography was performed on CIM QA monolithic columns (Sartorius BIA Separations), with immobilized positively charged quaternary amines. The quality of the obtained virus stock was assessed with SDS Page electrophoresis, followed by Western blot analysis. Finally, infectivity of recovered virus was evaluated by titration.

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2016

Influenza vaccines are still predominantly produced in embryonated chicken eggs and the purification processes barely have changed during the years. There is a growing need for fast, efficient and economical vaccine production.
So far, monolithic supports have been used successfully in virus purification and concentration, as well as in the purification of virus-like particles (VLP) propagated in cell cultures.
Therefore, our aim was to prove the applicability of monoliths in purification of influenza virus A propagated in embryonated chicken eggs.

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Downstream processing of viruses in virus vaccine or virus vector production accounts for up to 70% of the overall production costs. Immunoaffinity chromatography is a powerful purification technique due to its high specificity but is disadvantageous by the fact that the elution conditions are often detrimental for both the immobilized proteins and target antigens, especially viruses.
This application note describes the mumps virus purification using monolith-based immunoaffinity stationary phase and recently invented native elution of the bound viruses using amino acid solutions under physiological pH.
 

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2015

Determining the concentration of viruses is a crucial step in any production process. The most commonly used methods for virus quantification are either based on the infectivity of the virus (plaque assay, TCID50) determination of their genomic material (qPCR), or protein content (SRID, ELISA) and are very cumbersome and time consuming. HPLC analytical methods represent a fast alternative to these assays since they provide information on the virus content and purity in a matter of minutes. In addition to that, the data obtained is very reproducible and accurate.


For any kind of quantification, a calibration curve obtained with a virus standard is needed. The work presented in this application note shows the excellent performance of the CIMac™ Adeno Analytical Column – a monolith based anion exchange column, designed for fast and reproducible analyses of adenoviruses.

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Adeno-Associated Virus (AAV)-based vectors of various serotypes are considered to have high potential in human gene therapy and genetic vaccination applications. During manufacturing of AAV vectors undesired, incomplete particles are co-produced. They lack recombinant viral genomes and consist of empty capsid proteins only. Empty capsids increase the required dose of AAV virus for medical applications and are thought to cause immunological reactions against the vector capsid, leading to unwanted side effects. Removal of empty capsids during manufacturing, as well as the ability to quantify the amount of empty AAV particle content in a formulation is hence a critical requirement for any AAV production process.


Current methods for preparative separation of empty capsids (CsCl or iodixanol gradients) are challenging to scale-up and are not suitable for large-scale production. Furthermore, analytical methods for detection of empty capsids and determination of full to empty particle ratio (electron microscope (EM) assay, total particle assay [ELISA] combined with genome copy titration [qPCR]) are time- and labour consuming, influenced by operator technique or do not provide readily available reagents for different serotypes of AAV.


A new approach for separation of full and empty AAV8 particles by exploiting minor charge differences is presented in this application note. By using linear gradient elution on a CIM QA Disk Monolithic Column, a simple, rapid and reproducible assay for analysis of AAV particles is introduced. The method was successfully applied to AAV8 particles prepared by two different manufacturing processes.

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Adeno-associated virus (AAV) vectors of various serotypes are considered to have high potential for gene therapy applications. Currently, manufacturing of AAV vectors faces the challenge of co-production of incompletely formed particles lacking a recombinant viral genome. Empty capsids increase the dose of total AAV administered for efficient transduction and are thought to cause unwanted immunological reactions against the virus. Removal of empty capsids during manufacturing, as well as analysis of empty/full AAV particle content is therefore a critical requirement for any AAV production process. This Application Note demonstrates how CIMmultus QA monolithic columns can be used to remove empty AAV capsids from the product chromatographically in a single step.

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2014

Orthoreoviruses are dsRNA, non-enveloped viruses that can cause severe enteric and respiratory infections in humans and other animals. It is speculated that these viruses might be an important zoonotic pathogen. As such, orthoreoviruses can cause infections of undetermined etiology which are difficult to resolve. Next-generation sequencing (NGS) is a new technology which enables gathering a huge amount of genomic information from a sample in a short period of time. NGS is being increasingly applied in animal screenings for pathogen discovery and has a great potential in clinical microbiological diagnostics. However, the preparation of high-quality and high-quantity nucleic acid samples is a major concern for efficient application of the method.


CIM QA® disk in combination with NGS was used for discovering a novel reovirus in stool samples of a child with gastroenteritis infection of undetermined etiology. Two different starting samples were compared: clarified stool suspension and supernatant from cell culture inoculated with clarified stool suspension.

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2013

One of the most important plant viruses causing great economical losses in potato production is the filamentous Potato virus Y (PVY); virion size is 740 nm × 11 nm. Preparation of the pure virus suspension is essential for in vitro characterisation of the virus and also in many applications (e.g. antibody production). Virus purification usually consists of complicated and time-consuming protocols involving several ultracentrifugation steps, which are needed for isolation of the virus from the complex plant tissue matrix.


Different column chemistries, mobile phases and sample preparation strategies were examined during the method development study. Based on the obtained results, an optimised purification method for PVY from plant tissue on a CIM® QA Disk Monolithic Column was designed. The presence of the virus in the chromatographic fractions was monitored with viral RNA quantitation (RT-qPCR), viral protein detection (SDS-PAGE) and observation of the viral particle integrity (transmission electron microscopy).

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2012

Environmental water is contaminated with human enteric viruses through the discharge of sewage contaminated water. As a consequence, they are present in various environmental water sources: irrigation water, wastewater, recreational water, ground or subsurface water, and even drinking water. The continuous low level transmission of these viruses can result in the spread of some viral infections. The nature of most enteric virus diseases is such that they elude epidemiological studies. Improved detection of viruses that are present in low concentration could prevent a considerable number of infections. Among the most important human food-borne viruses are Noroviruses (NoVs), members of Caliciviridae family and hepatitis A virus (HAV) which can be the source of serious outbreaks.

CIM® monolithic columns in combination with ultracentrifugation and RT-qPCR were used for the concentration and detection of hepatitis A virus and feline caliciviruse, a norovirus surrogate. At the same time efficiency of newly developed method was compared with reference method, based on membrane filter.

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2011

Virus like particles (VLPs) are particles that structurally resemble viruses, but do not contain any genetic material. They are formed when structural viral proteins spontaneously self-assemble in transfected cells. Extracts from expressing cells contain not only VLPs, but also cellular DNA and proteins. These need to be removed in order to obtain pure VLPs, which are then applied for the production of vaccines, as delivery systems, as well as in other fields of nanotechnology applications (for the application on DSP of Ad3 VLPs check the Application Note A029). The purity of the final VLPs product is evaluated by methods like SDS-PAGE, agarose electrophoresis, PicoGreen analysis, BCA or Bradford assay.

In this work, CIMac™ QA Analytical Column was used for in-process control of the adenovirus serotype 3 dodecahedric virus-like particles (Ad3 VLPs). Samples obtained from different purification steps were injected on the CIMac™ QA Analytical Column and elution profiles were compared.

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Virus like particles (VLPs) are particles that structurally resemble viruses but do not contain any genetic material. They are formed when structural viral proteins spontaneously self-assemble in transfected cells. After VLPs are formed they need to be purified. Since the extract from expressing cells contains not only VLPs but also cellular DNA and proteins, VLPs purification represents a great challenge for the downstream processing.

Adenovirus serotype 3 dodecahedric virus-like particles (Ad3 VLPs) are an efficient vector for the delivery of the anticancer antibiotic drug bleomycin (BLM) – the use of Ad3 VLPs results in over 100 fold improvement of BLM bioavaliability. Ad3 VLPs are formed from penton bases of the adenovirus serotype 3 (Ad3) after these penton bases are expressed in a baculovirus/insect cell system. Ad3 VLPs are approximately 28 nm in size and have a molecular mass of 3.6 MDa. The current purification process of Ad3 VLPs consists of two purification steps, ultracentrifugation with a sucrose gradient (step 1) and ion-exchange chromatography (step 2) on Q-Sepharose and the whole procedure takes 5 days. Since Ad3 VLPs are large biomolecules, monolithic technology was applied for their purification with the aim to speed up the purification process.

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2010

Adenoviruses are among the most commonly used vectors for the delivery of genetic material into human cells and as such there is demand for high-titre manufacturing processes. The key to the successful development of such processes are analytical methods that can be applied to the final purified samples and throughout the production process. Many conventional methods for quantitative analysis of adenoviruses are labour and time-intensive. For example, a plaque assay can take up to 7 days to perform, is prone to error and will only report the number of infectious and not total viral particles. The resolving power of the high-performance liquid chromatography (HPLC), on the other hand, permits separation of intact virus particles from other cellular contaminants or virus particle fragments.


Anion-exchange chromatography has already been applied to analyse various adenovirus preparations. The results from the anion-exchange HPLC methods can be obtained much faster, within minutes, thus allowing for a faster evaluation of different process steps. A method was designed and developed to quantify adenoviral particles using a strong anion-exchange CIMac™ Analytical column. Regeneration conditions were incorporated to extend the functional life of the column.

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Adenovirus vectors have proven as useful tool for gene therapy, vaccine therapy and basic biology studies. The increasing importance of the recombinant adenoviruses pushes the limits of research in the field of adenovirus purification methods. There is a global focus on large scale production of adenovirus vectors, providing high titres combined with fast, effective and reliable purification methods.


Because of the physico-chemical properties adenovirus vectors possess, they can effectively be purified using ion-exchange chromatography. Here we present a simple and rapid method for adenovirus vectors purification using ion-exchange CIM ®QA chromatographic supports (Figure 1). CIM® monolithic supports are a new generation of chromatographic supports able to meet the GMP and GLP requirements in the field of virus purification.

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