Enrichment of phosphopeptides prior to LC-MS analysis is a crucial sample preparation step because of their low stoichiometry in biological sample, longer retention on reversed phase columns, and lower ionization efficiency compared to non-phosphorylated peptides .The use of metal oxides, most prominently of TiO2 enabled efficient and relatively simple phosphopeptide-enrichment. In this study a new monolithic column from BIA Separations containing immobilized TiO2-nanoparticles was tested for its ability to enrich phosphopeptides. The TiO2-column was also tested for possible carryover originating from biological samples. In conclusion, tested monolithic TiO2 columns show significant binding ability for phosphopeptides and are considered as suitable for phosphopeptide enrichment.
The demand for human immunoglobulin is invariably increasing on an annual basis. To satisfy demands, different manufacturing processes are used to isolate immunoglobulins from human plasma. A quest for alternative paths in manufacturing not only requires development of the most economical manufacturing process, but also a rapid method development and development of reliable analytics for manufacturing monitoring. For an efficient improvement of the purification methods as well as for in-process control during manufacturing stage, the usage of reliable and fast analytical techniques are of crucial importance.
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. 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 during the IgG production from Cohn I + II + III paste, however, a very accurate qualitative information about the composition of the sample can be obtained in less than 5 minutes.
In recent years bacteriophages were identified as a useful potential tool for different applications such as alternative to antibiotics, detection of pathogenic bacteria, delivery vehicles for protein and DNA vaccines and as gene therapy delivery vehicles. 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 CsCl2 density gradient ultracentrifugation, which is however difficult to be scaled-up. An alternative is chromatography, which already proved to be efficient for separation and purification of certain virus types. Methacrylate monoliths (CIM Convective Interaction Media® monolithic columns) were designed for purification of bionanoparticles and they already proved to be very efficient for concentration and purification of several plant and human viruses (influenza A, influenza B, adenovirus type 5, hepatitis A and others).
Our aim was to investigate whether CIM methacrylate monolithic columns can be implemented for purification of phages. Staphylococcus aureus phage VDX-10 was selected. Chromatographic support chemistry and buffer screening led to development of purification method on strong anion exchanger. Optimised single step purification method developed for S. aureus VDX-10 phage on CIM® QA monolithic column resulted in efficient removal of host cell DNA and proteins with high recovery of viable phage.
The challenge of efficient purification of gene therapy vectors
• The most commonly used gene transfer vectors are adenoviruses, lentiviruses, adeno-associated viruses, retroviruses, vaccinia viruses, and pDNA
• Due to their large size and sensitivity to pH, temperature and shear stress, purification is challenging and time-consuming
• A fast and efficient downstream processing purification method is required to isolate sufficient amounts of vectors with the final purity and state that conforms to stringent regulatory demands.
Solution: Convective Interaction Media Monoliths
• Convective interaction media (CIM) monolith chromatography
• Functionalised polydimethacrylate (QA, DEAE, OH, SO3)
• Precisely defined pore sizes
• Radial flow of solute
• Convective mass transfer
Challenges in monitoring the quality of vaccine production
• Process Analytical Technology (PAT) ensures process reproducibility in bioprocessing
• A mechanism to design, analyze and control pharmaceutical manufacturing processes through the measurement of critical process parameters (CPP) which affect product quality attributes (CQA)
• Initiated by the FDA as part of the 21st Century GMP initiative in 2001 with the goal of increasing productivity
• Application of PAT in vaccine development and manufacturing is challenging due to the sample complexity and batch-to-batch variability.
• During the development of an up- and/or down-stream process of the target biomolecule, a fast, accurate and reliable analytical method is requried for determining the quantity and purity of the product intended for human use
Solution: Convective Interaction Media Monoliths
• Monoliths are chromatography media cast as a single block, inserted into a housing
• Highly inter-connected network of channels (1-2 μm) containing functionalised binding sites for large biomolecules (viruses, VLPs, pDNA, antibodies)
• Performance unaffected by increasing the flow rate or molecular size
The development of safe, effective, and affordable vaccines has become a global effort due to its vast impact on overall world health conditions. A brief overview of cancer vaccine characterization techniques, especially in the area of high-resolution mass spectrometry, is presented. It is highly conceivable that the proper use of advanced technologies such mass spectrometry, along with the appropriate chemical and physical property evaluations, will yield tremendous in-depth scientific understanding for the characterization of vaccines in various stages of the development. This work presents the physiochemical and biological characterization of two cancer vaccines: Racotumomab and Her1-ECD. Racotumomab monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. The Her1-ECD is a vaccine preparation based on the extracellular domain of HER1 and it is being evaluated in Phase I clinical study in patients with refractory prostate cancer.
A monolith is a stationary phase made of single piece of porous material. Unlike conventional particle-shaped chromatographic supports, the pores of the monolith are interconnected and form a network of channels with diameters ranging around 1500 nm. The binding sites in these channels are highly accessible for target molecules and since the predominant mass transfer depends on convection rather than diffusion, the dynamic binding capacity is flow independent. These characteristics make the monolithic supports suitable for fast separation and purification of large biomolecules such as proteins, DNA and viruses, which sometimes exceed 200 nm in size and thus have low diffusion constants.
In this work we tried to quantify influenza A virus using an analytical CIM monolith column. First a screening of available CIM stationary phases was performed in order to establish the optimal stationary phase for the binding of the virus. The effect of the mobile phase composition and pH on the recovery and peak shape of the virus was investigated. Linearity was examined. The amount of virus in the flow-through and elution fractions was determined with the haemagglutination assay and the purity of the fractions with SDS PAGE. All experiments were performed with an inactivated Influenza A/Wisconsin PZC whole virus sample that was produced in eggs.
There are two different designs of chromatographic columns concerning the flow profile. Most of today's HPLC columns belong to the group of so-called axial mode operating columns, while the radial ones with a radial flow pattern are more rare. Which type performs better depends on the particular case but it seems that the radial operating columns are attracting interest since they exhibit some beneficial features. One of the main problems of radial operating chromatographic columns is the changing of a mobile phase linear velocity over the chromatographic bed. Because of that, matrix efficiency for porous particulate supports varies by its position within the bed, and overall performance is more difficult to predict.
This problem is not present when the monolithic supports are used, since it was demonstrated that their chromatographic properties are flow unaffected even at the extreme linear velocities. This was confirmed also for the radial operating mode.
The monolith and radial flow housing were designed for extremely high flow rates, up to 70 CV/min, which is the range of the flow rates applied on membranes. This was achieved by proper monolith dimensions with the height of 55 mm, inner diameter of 6.0 mm and thickness of only 4.5 mm.
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.
Recombinant Adenovirus (rAd) is commonly used for vaccination and gene transfer for cancer applications. This vector is widely used in phase I/II clinical trials. Therefore we believe that upstream and downstream processes should be improved.
We developed a production manufacturing process for rAd serotype 5 n HEK293 grown into disposable fixed-bed iCELLis™ bioreactors (ATMI LifeSciences). The purification process was reduced to one single chromatography step using the Convective Interaction Media, anion exchanger (CIM ® QA monolithic column, Bia Separations).
Briefly, rAd particles were extracted from cells using Triton X-100, depth filtered to discard cell debris, captured and purified out on CIM ® QA. The shallow gradient used for the elution of the vector allowed the separation of different rAd particles populations more or less enriched in full particles. A final step based on Tangential Flow Filtration (TFF) in hollow fibers allowed the removal of remaining impurities and the formulation of the vector batch.
In addition, we developed an analytical method on CIMac™ QA analytical column (Bia Separations) to characterize the different steps of the process, and to track the differences linked to the production runs to increase the robustness of the process. This method provided elution profiles for each step as well as titer of the purified rAd in the final step.
The rAd was produced in an iCELLis™ nano fixed-bed bioreactor (0.5-5.3 m2), purified in a 8mL CIM ® QA monolithic column, scaled up in a medium-scale size 80mL column. We are currently extending the rAd production in a 133m2 iCELLis I000™ bioreactor with a purification step using a 8L CIM® QA monolithic column to purify out up to 1x1015 vector particles.
Monolithic supports represent a new generation of chromatographic media. Due to their large inner channel diameters and enhanced mass transfer characteristics, methacrylate monoliths (CIM® monolithic columns) offer efficient and fast separation of large biomolecules like pDNA, viruses and monoclonal antibodies. High binding capacity for viral particles, good product recovery and resolution are also benefits of monoliths. During loading of MDCK cell-derived H1N1 inactivated influenza virus particles onto monolithic columns, increased back pressure is sometimes observed. This is especially an issue if a large amount of virus needs to be purified since the back pressure depends on the loading volume. The goal of this work was to determine the factors contributing to this effect. We tried to prevent the increased back pressure by treating virus harvests with different precolumn phases (LRATM - Lipid removal agent, Amberlite® XAD 7HP, epoxy monolithic column) and by filtering the virus material before loading it onto the column. To compare different pre-treatment strategies of the virus material the dynamic binding capacity of CIMac QA for virus was first determined, resulting in approximately 1x1013 virus particles per ml. Than loadings of the pre-treated virus material at 75% of the column capacity were performed and mass balances for the virus, DNA and proteins were investigated. Another goal of this work was to find a good regeneration strategy for the columns where increased back pressure occurred. For this reason different regeneration procedures using lipase, benzonase, 2-propanol and NaOH treatment were tested on the columns with increased back pressure.
Traditional waste water treatment usually does not remove or inactivate all of the potentially pathogen microorganisms present in the waste water. This is especially true for enteric viruses that are introduced into the environment through the discharge of effluent from waste water treatment plants - WWTP (Simmons et al, 2011). Although discharged concentrations of viruses are low they can still lead to infection. For some enteric viruses ingestion of only 10 - 100 virus particles is enough to initiate the disease, what calls for very sensitive detection methods. It has been previously shown that CIM-quaternary amine (QA) monolithic supports are a good tool for concentration of viruses in water (Gutierrez-Aguirre et al, 2011). Here we go one step further and evaluate CIM monoliths not just for concentration of enteric viruses but also for their removal from effluent waters.
Potato spindle tuber viroid (PSTVd) is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and uncapsidated RNA molecule with 359 nucleotides and no coding capacity. Because of its complex secondary/tertiary structure it is very stable ex vivo and it is easily transmitted mechanically by contaminated hands, tools, machinery, etc. In this work, we describe the development and optimization of a method for concentrating PSTVd using CIM monolithic supports.
Objective – Influenza VLP
• Complex structure
• Different protein components
• Host cell derived lipid membrane
• ESAT6 epitope of M. tuberculosis engineered into influenza hemagglutinin [1,2]
• Optimal vaccine candidates
• Induce strong immune response 
• Contain no genetic information
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.