Monolith chromatography media coupled with metal affinity ligands proved superior to the conventional particle-based matrix as a plasmid DNA (pDNA) purification platform. By harnessing the differential affinity of pDNA, RNA. Host cell proteins and endotoxin to copper ions in the solution a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl2-induced precipitation. RNA and remaining endotoxin were subsequently processed by copper immobilized metal affinity column employing either monolith or particle-based matrix where both RNA and endotoxin were removed below detection limit with almost complete recovery of pDNA in the monolith was found to have several advantages in terms of handling feedstocks crowded with RNA in a concentration-independent manner and exhibiting flowrate-independent dynamic binding capacity for RNA. This enabled monolith-based process to be conducted at high feed concentration and flow rate. Resulting in pDNA vaccine purification at a high yield and purity and the process conditions investigated, the use of monolith column gave at least three fold higher productivity for recovery of purified pDNA as compared to the particle- based column, demonstrating its potential as a more rapid and economical platform for pDNA vaccine purification.
The present study describes a new methodology to quantify and monitor the quality of supercoiled (sc) plasmid DHA (pDLIA), using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows distinguishing the plasmid isoforms by a NaCl stepwise gradient. The selectivity, Linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a sc plasmid concentration up to 200 µg/mL. The main advance achieved by using this monolithic method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, in a 4 minutes experiment. This work also intends to evaluate the possibility to assess the sc pDNA present in more complex samples, allowing the control of the samples recovered from different bioprocess steps.
Glycosylation is one of nature mechanism for invreasing the diversity of protein structures affecting biophysical vjaracterostocs and bioactivity. Glycoproteins exist as mixture of different isoforms ("glycoforms"). In this mixture a group od different glyco components is attached to individual glycosylation site. Different glyco componets attached to the same site may have diggerent effect on biophysical charachteristics of glycoproteins. The type of glycosylation and the degree of heterogenity are important for many reasons starting from stability, activity, clinical efficency (toxicity, pharmacokinetics, immunogenicity), to standardization and patentability.
Thus, it is necessary to separate glycoforms and as much as possible to difine the heterogenity i.e. population of of glyco components attached to the singele glycosysilation site.
External invertase is a widely usef model for studying the influance of the glyco-component on protein stability. External invertase from yeast Sccharomyces cerevisiae has 14 potential N-glycosylation sites in the sequence, 13 of which are fully or partially glycosylated with olygomannans of varying sizes.
Extensive research in the last two decades has led to the realization of Immunoglobulin M (IgM) as a potential therapeutic and diagnostic agent for autoimmune diseases, infectious diseases and as an AIDS and cancer vaccine. Growing interest in these molecules has created a need for an accurate, rapid and simple analytical method to measure IgM concentrations during the production (in-process control) in cell culture supernatants as well as in all purification steps in the downstream processing.
Convective interaction media (CIM) monolithic columns has been increasingly recognized as a quantification tool for large molecules. Affinity ligands like protein A and protein G are the most common ligands used for antibody capture and analysis.
In the last few years pharmacology has made a big step towards the new type of drugs, called biological drugs. Popularity and market for biological drugs grew exponentially, so did the need for fast and inexpensive purification. Classic liquid chromatography columns were unable to separate biological compounds in industrial quantities, therefore the scientists were looking for alternatives. One of them are monolithic materials. Monolithic materials, especially methacrylate monoliths, are becoming more and more popular in separation processes due to their fast separations, low pressure drop and mechanical stability.
In the context of preparing new columns and improving existing ones, we need to know every single chemical as well as mechanical property of our monolithic material. Here we present some key data and interesting correlations between mechanical and structural properties of GMA-co-EDMA porous monolith. In the first paragraph we compare nonmodified and DEAE modified monoliths with different average pore size and porosity, regarding to their compression and tension properties. The second paragraph deals with the impact of these parameters on the permeability of the column during separation.
Commonly, epoxide-based monoliths used as porous supports in affinity chromatography are synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) by free radical polymerization.
We prepared an epoxide-based monolith by self polymerization of polyglycidyl ethers where the epoxy groups serve as functional groups for the polymerization reaction as well as for the immobilization of the ligand.
Monolith technology has been employed in chromatography for a variety of applications using diverse substrates. The development of different column chemistries has led to the Thermo Scientific ProSwift line of monolith columns for analytical protein separation by ion exchange and reversed phase. Separation of biomolecules can be achieved at elevated linear velocities with minimal loss of resolution. Columns are designed to withstand extreme pH cleaning, desired for sterilization. The backbone and functionalization are optimized for high mass loading for small-scale preparative applications, the ideal first dimension separation of crude biological samples. Combined with increased sensitivity of a 1 mm format, detection of proteins of very low copy number in a crude samples is achievable.
We discuss here the ability to produce highly-reproducible columns with excellent stability as well as characteristics required for fast small-scale preparative analysis. HPLC column selection is a challenging task, specifically where the mixture contents is somewhat unknown. Many factors influence the choice of column used; chemistry, robustness, and reproducibility. For quality assurance, columns should be chosen that are reproducible both run to run and batch to batch. To prevent cross-contamination between samples, carryover and sterilization should be considered. For semi-prep, a combination of high mass loading and good resolution enable increased purity of peak fractions. Format and operational flow rate should be considered with respect to multidimensional analysis.
There is an increasing demand for highly purified immunoglobulin G since they have found wide range of potential application in immunodiagnostics and immunotherapy.
Human IgG (hIgG) consists of four subclasses (IgG1, IgG2, IgG3 and IgG4) that show differences in some of their physicochemical characterictics and biological properties.
The present research project aims to separate subclasses of hIgG using monolithic stationary phase by SMB technology.
Application of plasmid DNA for gene therapy and vaccination has gained huge interest in last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Major influence on achieving regulatory demands in pDNA production has downstream processing and in order to get optimal purity different purification techniques have to be included. It was demonstrated that methacrylate monoliths can be used for efficient purification process of plasmid DNA. High dynamic binding capacities and high flow rates of methacrylate monolith enabled excelent purity and productivity.
Analysis of a large number of samples requires chromatographic support that not only enables fast separation and purification of a target biomolecule from a complex matrix but also support an automation of a process. The methacrylate 96-well monolithic plate format enables both. 96-well monolithic plate reduces experimental time because it allows fast and efficient evaluation of parameters for binding and elution conditions. This format is a quicker alternative to several consecutive tests on chromatographic column.
Protein L binds certain types of kappa light chains containing Fv and Fab fragments prepared from antibodies. In the case of IgG's the strong binding affinity refers only to human, mouse and rat species. It offers an advantage over Protein A and G as it binds to kappa light chains regardless of heavy chain subclass and can therefore binds up to 60% of IgG antibodies from human serum sample.
The main goal of our work was the preparation and characterization of CIM Protein L disks. First, Protein L disks with different densities of Protein L on the support were prepared in order to define the dependance of the IgG capacity on the amount of the bound Protein L. Further on, the method of characterization of Protein L disk using IgG was developed. In the end, the stability of the developed CIM Protein L disks in different solutions was tested in order to define the operating and storage conditions.
In recent decades much work has been done on the development and optimisation of chromatographic supports in order to achieve efficient purification of biomolecules.
In the presented study we have investigated hydrodynamic and chromatographic properties of weak anion-exchange grafted monoliths (DEAE). Varying the concentration of the grafted polymer, grafted monoliths with different layer thickness and degree of branching were obtained. This results in a different hydrodynamic and chromatographic behavior of the examined monoliths such as permeability, ionic capacity and dynamic binding capacity (DBC) for the BSA protein. The DBC increases with the grafted layer thickness probably due to higher number of binding sites available for binding of the macromolecules. However, longer chains contribute to the reduction of the pore volume which results in a higher pressure drop. The latter can be additionally increased when biomolecules of interest are bound to the matrix. From this data information about the penetration depth into the grafted layer can be obtained giving an insight into the binding mechanism. Since the flow-unaffected properties were preserved even for large biomolecules, grafted monoliths may become a resin of choice for downstream processing of various macromolecules.
CIMac™ Analytical Columns are high-performance monolithic columns offering all the advantages of a special continuous short polymeric bed and are primarily intended for fast, efficient and reproducible separations of biomolecules like large proteins – antibodies (IgG, IgM), plasmid DNA, phages and viral particles. Their small volume and short column length allow the operation at high volumetric flow rates (from 1 to 30 column volumes/min) thus enabling receiving the information about the product quantity and purity in just a few minutes. These columns are pre-packed in dedicated stainless steel housings and allow user friendly connections to HPLC equipment. The product family offers strong cation exchange, strong and weak anion exchange and specialty analytical column for plasmid DNA. All columns can be effectively used for the in-process and final control of various samples from different purification process steps.
The biotechnological production of recombinants proteins consists of two main processes, upstream (biosynthesis) and downstream (protein purification) process. During the last decades the upstream process for mammalian cell culture has been improved significantly yielding in high amounts of protein. This development however led to a new challenge : the downstream process became a bottle-neck because of the large amounts of protein per batch in combination with the protein specific behaviors at high concentration.
In protein purification preparative chromatography is synonymous to “column chromatography”, and the favorable statics of a column are out of question for the physical requirements of beaded matrices. However, when approaching larger scales the physical dimensions of chromatography columns turn unfavorable: shallow gel beds of wide diameters. The footprint of such device increases drastically as does the weight, consequently resulting in limitations regarding floor space and floor bearing force.
A suitable chromatographic base matrix that is not obliged to a distinctive column design is a single piece of polymer – a monolith. Leaving the conventional column design, we have constructed a device for a monolith of rectangular shape, with the size of the monolith only limited by total weight (e.g. for handling and / or transportation). Using this design in a modular way, the individual modules can be stacked to make use of the height of a room at a very low footprint. A specific distribution system for feeding the monolith modules has been designed to allow a true linear scale-up from laboratory to large technical scale.
Application of plasmid DNA for gene therapy and vaccination has gained substantial interest in the last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Downstream processing has major influence on achieving regulatory demands in pDNA production and in order to get optimal purity different purification techniques have to be applied. It was demonstrated that methacrylate monoliths can be used for efficient purification process of plasmid DNA. High dynamic binding capacities and high flow rates of methacrylate monolith enable excellent purity and productivity.
Bacteriophages were in recent years identified as a useful potential tool for different biotechnological applications such as alternative to antibiotics, detection of pathogenic bacteria, delivery vehicles for protein and DNA vaccines and as gene therapy delivery vehicles (1). For all listed fields of use it is important that phages are highly purified with preserved biological activity. Phage and other virus purification have traditionally been carried out by CsCl density gradient ultracentrifugation, which is however difficult to be scaled-up. An alternative is chromatography already proved to be efficient for purification and concentration of certain virus types.
One of the key issues using chromatography is processing time and capacity of the resin. Novel type of chromatographic resin named monoliths was already proved to be very efficient for fast separation and purification of macromolecules as are large proteins, DNA and viruses (2,3,4).
Our aim was to investigate whether Convective Interaction Media (CIM) methacrylate monolithic columns can be implemented for purification and concentration of phage T4 (virus for E.coli). Chromatographic method using linear gradient was implemented to investigate conditions for phage elution and to establish the optimized chromatographic method applying step gradient. We analyzed phage recovery and purity together with method reproducibility.
Anion-exchange chromatography is fundamental in downstream processing of plasmids both as a process and analytical technique. CIM anion-exchange monolithic columns have already been successfully used for the industrial scale purification of pharmaceutical grade small plasmid DNA .
In this work we report about the use of the newly developed monolithic analytical column intended for plasmid DNA determination in terms of its analytical performance. Higher degree of sensitivity, precision and accuracy is necessary in order to determine the quality of clinical grade DNA intended for therapeutic use. Plasmids purified from Escherichia coli fermentation exist predominantly in the supercoiled form (SC) the other two topoisomers present in the final product are mostly the open circular (OC) and linear forms . Different chromatographic conditions were tested and the separation was optimized in terms of buffer and pH selection as well as in terms of gradient slope and column length. The results were compared to the results obtained with established analytical methods.
A number of IgM monoclonal antibodies are currently in development for treatment of autoimmune disease, infectious disease, and cancer. Growing interest in these molecules has created a need for an accurate, rapid, simple analytical method to measure IgM levels in cell culture supernatants, and to document the distribution of IgM and protein contaminants in chromatography fractions. High performance protein A columns are used for this application with IgG monoclonals, but IgMs are easily denatured by the harsh conditions required for elution of most affinity ligands. However, IgM monoclonals often exhibit strong retention on either cation exchangers, or anion exchangers, or both, making ion exchange chromatography a potential candidate for this application.
The large size of IgMs makes them a major challenge to particle-based chromatography media. Pentameric IgM has a mass of about 0.96 Md, and hexameric IgM about 1.15 Md. Their diffusion constants are about 2.5 x10-7 cm2/sec, about twice as slow as IgG. Since particle-based chromatography media mostly rely on diffusion for mass transport, both resolution and capacity are im- Figure 4 illustrates a modified anion exchange gradient configuration for monitoring the amount of IgM expressed in cell culture supernatants. A wash step was introduced to better remove con- paired, and increasingly so at higher flow rates.
Monolithic ion exchangers are characterized by an interconnected system of channels with diameters ranging 0.5 to 2.0 microns. This pore architecture supports convective flow, which conserves high resolution at high flow rates. The lack of a void volume removes the major source of dispersion in chromatographic systems. This contributes to sharper peaks, which improves both resolution and sensitivity. Capacity is also conserved at high flow rates. This permits use of a microcolumn format that minimizes assay time and buffer consumption. This combination of features should make monoliths effective analytical tools for IgM.
Membrane based anion exchangers are being used increasingly for purification of monoclonal antibodies. The transition from particle-based anion exchangers is driven partly by the convenience of membranes and partly by the cost saving associated with their disposability, however the feature that makes them functionally superior is more effective mass transport.
IgM can be used for several purposes such as early detection of certain diseases or, when labelled, localized cancer tumours. For their purification commonly chromatography is used. Methods for purifying such big molecules (M.w. around 950 kDa) are usually long and time consuming since these molecules have extremely low mobility therefore mass transfer between mobile and stationary phases is significantly reduced. When purified using affinity mode, serious decrease in IgM activity can occur because of long exposure to low pH in which they are unstable. Furthermore, because of their size, the IgM capacity of convenctional resins is rather low. CIM monoliths were already successfully used for fast separation of large molecules. In this work we tested applicability of anion-exchange CIM monolithic columns for preparation of IgM.