PEGylation involves the formation of a stable covalent bond between activated poly (ethylene glycol) polymers and polypeptidic drugs and molecules. This process causes a change in protein hydrophobicity and results in variance between the obtained conjugates. Despite this, hydrophobic interaction chromatography (HIC) is used less frequently for separation of PEGylation reaction products than other techniques. Separation of PEGylated conjugates of Ribonuclease A (RNase A) via HIC on monolithic supports was analysed in this work. The protein was PEGylated in the N-terminal amino group with 20 kDa methoxy poly (ethylene glycol) propionaldehyde.
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).
Transferrin (Tf) is a glycoprotein that transports iron to cells and has two N-glycosylation sites in humans – at asparagine 432 and asparagine 630. Carbohydrate-deficient Tf, which lacks one or both N-glycans, is the most common marker for congenital disorders of glycosylation.1 Altered Tf glycosylation has also been reported in hepatocellular carcinoma2 and chronic alcohol consumption.3,4 High-throughput Tf purification and glycan characterisation methods are under extensive development in order to facilitate screening of glycosylation patterns for population, genetic and clinical studies.
This application note describes the development of an immunoaffinity purification method on a CIMac™ analytical column with immobilised anti-transferrin antibodies (@Tf) and the successful transfer of the method to the monolithic 96-well plate (CIM® @Tf-0.2 monolithic 96-well plate). The affinity purification method has been used for Tf isolation from human blood plasma followed by ultra-performance liquid chromatography (UPLC) analysis of Tf N-glycosylation.
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.
CIMac™ r-Protein A Analytical Column is a short bed, high performance monolithic column. It is intended for fast, efficient, and reproducible qualitative and quantitative analyses of Immunoglobulin G (IgG) and suitable for use with HPLC and UPLC systems. Quantification of IgG is possible between 0.2 μg and 20 μg. Its small volume and short column length allow operation at high volumetric flow rates (up to 3 mL/min). The information about product quantity and purity is thus generated in just 1 minute! The column has an innovative symmetric design for bi-directional flow contributing to longer lifetime.
The CIMacTM antibody immobilization platform enables an elegant immobilization of antibodies, which can be used as immunosorbents in specific diagnostic applications as well as in downstream processes. In this work we show the dependence of the coupling strategy on CIM monolith with the chromatographic efficiency of final immunoaffinity adsorbent. Different activation chemistries were tested for the immobilization of two model monoclonal antibodies (mAbs) with subsequent chromatographic characterization of the affinity support.
Column used for this application note were CIMac CDI, AE, EDA, HDZ, rpA.
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.
The CIMac™ antibody immobilisation platform enables an elegant immobilisation of antibodies, which can be used as immunoaffinity adsorbents in specific diagnostic applications. Immobilization of antibodies to solid chromatographic supports is avery elegant solution for preparation of reusable immunoaffinity assays that can lower the price but also increase the accuracy and lower the limit of detection of biological assays for diagnostic purposes. Therefore, it is important that reusing a column for multiple sample analysis does not affect chromatographic/analysis performance. In this application note, an example of reusability of the hydrazide-based CIMac™ column ( HDZ ) with immobilised monoclonal antifibrinogen for consecutive purification of fibrinogen from human plasma is shown.
In diagnostic applications, fibrinogen is measured with clotting activity assay variations, together with other blood clotting factors. In research and preparative applications, larger quantities of anti-coagulants, such as EDTA and citrate, or coagulants such as Ca2+ are present in the samples, preventing successful and/or accurate fibrinogen concentration measurements. In such cases an alternative measurement method is needed.
The CIMac™ antibody immobilisation platform provides an elegant solution for immobilisation of antibodies, which are used as immunoaffinity adsorbents. It has been shown that several analytical runs can be performed on a single column. This simplicity is also combined with a fast method, where a chromatographic analysis can be completed in 4 min. Used column was hydrazide ( HDZ )-based CIMac™ column.
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.
The demand for human normal immunoglobulin is invariably increasing on an annual basis. To satisfy increasing demands, different manufacturing processes are used to isolate immunoglobulins from human plasma. 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 an efficient development of the purification methods as well as for in-process control during manufacturing stage, the uses of reliable and fast analytical techniques are crucial.
Fast and reliable fingerprint-based method for characterization of immunoglobulin G (IgG) prepared from Cohn I+II+III paste in two chromatographic steps is presented here. The fingerprint method bases on partial separation of proteins in linear gradient on CIMac™ QA 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples, however a very accurate qualitative information about the composition of the sample being analyzed can be obtained in less than 5 minutes.
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).
Process Analytical Technology (PAT) is of crucial importance in the process of IgM manufacturing, especially in its optimization where fast and reliable analytical methods capable of quantitation of the corresponding recombinant IgM concentration levels in the upstream processes are required.
Convective Interaction Media CIM® strong anion exchange monolithic columns have a great advantage in comparison to particle related methods due to their separation capability based on the convective flow mechanism that proved to be particularly efficient in the separation of large IgM molecules.
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.