Application Notes

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).

Lab scale production of recombinant human monoclonal antibodies (mAbs) is required for the identification and characterization of lead clones with potential therapeutic value. For this purpose, many mAbs need to be screened. MAbs titers in this type of production scale tend to be quite low (from 0.01 – to 0.1 mg/mL), therefore a substantial amount of material needs to be processed to obtain the right amount of purified mAbs. Speed of processing and the ability to capture mAbs from diluted harvest stock are essential in this type of mAbs purification.

In this application note, a quick purification procedure using a CIM® r-Protein A-80 Tube Monolithic Column that generated up to 100 mg of mAbs with a purity of more than 95 % is described. Elution of mAbs is performed using a two-dimensional gradient (pH 7.2 to 2.5; NaCl 150 to 500 mM), allowing gentle elution of a wide range of mAbs at moderate pH (pH ~4) without any method optimization. Using this procedure, approximately 30 different mAbs were purified, processing up to 5 L of loading material (2 times diluted clarified harvest).

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.

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.

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.

The demand for monoclonal antibodies is invariably increasing on an annual basis. To satisfy increasing demands, faster and cheaper ways of manufacturing are explored. A quest for alternative paths in manufacturing not only requires development of most economical manufacturing process, but also rapid method development and development of good analytics for monitoring of manufacturing. For a quickly developed process, the use of reliable and fast analytical techniques are crucial. Moreover, this analytical technique should than be preferably used also for in-process control during manufacturing stage.

Here we present fast and reliable method for processing and analyzing IgG, IgA ang IgM using CIM® QA Disk Monolithic Column, which thrive upon speed, repeatability and high capacity.

Diluted samples of live attenuated measles and mumps virus were each loaded on CIM® DEAE Disk. Concentrated eluates of viral RNA were subjected to molecular detection by PCR. It was demonstrated that enrichment of viral RNA on a CIM® DEAE Disk prior PCR is feasible and successful.

A Hemoglobin A1c reference standard was loaded on CIM® SO3 monolithic column and eluted in a mixed stepwise and linear gradient. HbA1a, HbA1b and HbA0 variants were separated and a complete determination of HbA1c (including equilibration) was obtained within 1.1 minute.

A mixture of IgG, HSA and IgM standard was loaded on CIM® EDA Disk and eluted in linear salt gradient at a flow rate of 4 mL/min (12 CV/min). A complete separation of IgM from IgG and HSA was obtained within 1.5 minute.

Immunoaffinity columns were prepared by immobilization of Protein G on CIM® Epoxy Disk, CIM® Epoxy tube (1 mL) and an activated, particle based agarose support. A comparison of productivity was performed by loading centrifuged human plasma and resulted in superior productivity of CIM® monolithic supports.