Miniaturised immobilised enzymatic reactors can be used for small scale digestion of proteins. There is need for such devices; small scale devices are used either for processing of analytical sample quantities, or as proof of concept before protein digestion at larger scale. This application note compares the performance of a flow through miniaturised immobilised enzymatic reactor (μIMER) with in-solution batch digestion of simple proteins and complex matrices. Automation of peptide analysis by coupled LC-MS is explored as an option to increase throughput. In the cases evaluated, the miniaturised immobilised enzymatic reactor offered comparative results to overnight in-solution digestion, within less than 10 minutes.
Pre-activated CIMmic™ monolithic columns with 100 μL bed volume were immobilised with trypsin from bovine pancreas. This small format allows coupling to HPLC for on-line protein digestion, as well as syringe (manual) operation of the IMER. Pre-treated samples (denatured, alkylated and ultra-filtered) are injected into the column, and the eluate (tryptic digests) are subjected to LC-ESI-MS-MS analysis for protein identification and post-translational modification (PTM) determination.
Coupling trypsin enzyme onto chromatographic supports provides a platform to reuse the enzyme and automate the hydrolysis process. A monolithic chromatographic support, such as Convective Interaction Media (CIM®), enables mass transfer of molecules within its channels exclusively by convective flow. This results in enzymatic conversion which is not limited by diffusion, making CIM® monoliths ideal for the preparation of immobilised monolith enzymatic reactors (IMERs). BIA Separations offers CIMac™ trypsin IMER with a bed volume of 0.1 mL as analytical platform for mass spectrometry (MS)-based proteomics. Larger volume IMERs (up to 80 mL) are available for industrial scale production of protein hydrolysates. The following example describes the enzymatic production of β-Lactoglobulin (β-Lg) hydrolysates using monoliths with 2 μm (N1) or 6 μm (N2) average channel diameter.
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 (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.
Sample displacement chromatography exploits the different relative binding affinities of components in a sample mixture to achieve accummulation of a desired substance on the column before elution. In pharmaceutical applications, requirements for purity and efficacy of plasmid DNA (pDNA) as a therapeutic product are stringent. The separation of linear, supercoiled (sc) and open-circular (oc) pDNA isoforms has already been established on CIM® butyl (C4 HLD) monolithic columns at preprative scale. This process requires high concentration of ammonium sulphate for loading which increases the overall production requirements. Competing adsorption in sample displacement chromatography utilises the binding capacity of the chromatographic resin more efficiently and increases productivity of the chromatographic step.
This application note investigates three monolithic chromatographic supports with different hydrophobicities regarding their applicability for sample displacement of pDNA. CIMac™ C4 HLD (butyl, high ligand density) as a commercial product and pyridine and histamine as custom immobilised columns are compared.
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.
Plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy and often one or more chromatographic steps are used in the downstream process. High ligand density butyl-modified chromatographic monolith (CIMmultus™ C4 HLD, part of CIMmultus™ HiP² Plasmid Process Pack™ 1-1, product number 100.0011-2) is currently used in a polishing step of a pDNA purification process (1), is mainly used for separation of supercoiled (sc) pDNA separation from open circular (oc) and linear pDNA isoforms as well as for removal of remaining gDNA and RNA.
This application note presents a comparison of two different polishing processes employing monoliths, namely bind-elute (BE) and the more recently described (2) sample displacement purification (SDP).
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.
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.
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.
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.
DNA immunization can potentially induce both, humoral and cellular immune responses, and thus comprises an attractive approach for the development of an effective vaccine against HCV. The pIDKE2 plasmid is the main component of the CIGB's candidate vaccine against Hepatitis C virus (HVC), which is being used in HCV chronically-infected individuals during clinical trials phase 1 and 2.
In order to satisfy the high demanding plasmids consumption for clinical trials, the downstream process was improved to reach the quantities need it for clinical trials.
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.
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).
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.
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.
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.
Filamentous phage M13 is a rod shaped non-lytic bacterial virus. M13 genetic material is used for many recombinant DNA processes, and the virus has also been studied for its uses in nanostructures and nanotechnology. The phage has been intensively studied for purposes of phage display and as a delivery vehicle for gene therapy. Phage display was first demonstrated with M13 bacteriophages and the filamentous phage remains a workhorse for this technology. Because of its typical size and rod shape it is considered as a challenging for purification. With large and highly interconnected pores monolithic chromatographic supports are also bridging that problem.
The ability to improve the purification process of M13 and other phages can have a significant impact on the market. By using phages for gene therapy, there will be a decrease in manufacturing time and production costs while enhancing the gene insertion. For phage display, a quicker method for phage purification will allow this powerful tool, which shortens the new drug discovery path and illuminates the basic interactions between different proteins, to be used with higher frequency.
Bacteriophages are used in a broad range of applications, including phage therapy and phage display. With the growing problem of antibiotic resistance leading to untreatable bacterial infections, they are becoming very interesting as antimicrobial agents, not only in medicine, but also in veterinary medicine, food industry and agriculture. Phages intended for use as antimicrobial agents, especially those for human use, need to be purified of contaminants.
Here we present efficient single step purification method for a Staphylococcus aureus phage VDX-10 from bacterial lysate on a CIM® QA Disk Monolithic Column (Figure 1). The described method can be used also on a larger scale using a CIM® QA-8 mL Tube Monolithic Column (Figure 2).
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.
Bacteriophages, viruses that infect bacteria, are being used as antibacterial agents, in phage display screening, as gene therapy delivery systems, and for bacteria typing. To use phages in these applications, they must be free of all impurities. A purification and concentration process was recently developed using an ion exchange monolithic column . One of the key challenges faced in phage purification is the monitoring of genomic DNA (gDNA) released to the growth medium which can interfere with the various applications of phages. CIMac™ DEAE Analytical Columns can be used to monitor the fermentation process, evaluate the amount of degraded gDNA to determine the optimal fermentation endpoint and then to efficiently purify the phage particles.