One of the handicaps of working with bacteriophages is the long duration required to perform plaque assays. Plaque assays also impose questions about accuracy and precision relative to the scale and experience of the persons performing and interpreting them. This poster presents a pair of high precision, high accuracy chromatography-based assays that permit determination of phage concentration in less than 1 hour. Sensitivity of UV absorbance is poor because of the low concentration of phages. However, phage sensitivity is strongly amplified by monitoring the chromatogram with either fluorescence or MALS. Fluorescence works by measuring the fluorescence emission from tryptophan residues of the phage proteins. MALS works by passing a laser beam through the sample and reading the scatter produced when it encounters a particle. Larger species generate more scatter.
Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants. The most common technique for purification is ultracentrifugation using cesium chloride gradients. This technique is elaborate, cumbersome, expensive and difficult to scale-up.
Alternative techniques for purification are usually time consuming and affect phage recovery and/or viability. In this study we present efficient two-step chromatographic purification method with binding phages to a stationary phase - Convective Interaction Media (CIM®) monoliths. The aim of the study was to develop robust, fast and effective virus purification platform that can be used for several types of bacteriophages for any application. In this work bacterial lysate with bacteriophage T4 (host E.Coli) was used.
Production of high value biological therapeutics usually involves complex manufacturing processes with high process variability. Additionally, development of robust and reliable bioprocesses can be challenging. PAT aims to enhance bioprocess understanding and implies a holistic approach to ensure that quality is built into products by design. Efficient PAT therefore calls for fast and robust analytical techniques which enables to asses high quality information about critical quality attributes and key performance indicators as parallel as possible to the manufacturing process. PATfinder™ is unique HPLC system for routine gradient separations that enables every analytical task. Equipped with bio-inert ceramic pump heads is deliberately tailored to meet the demands of analytical applications covering wide range of biomolecules. Highly sensitive and fast multi-wavelength detector enables to detect component peaks even in very fast gradients.
Productivity of the downstream bioprocessing depends among others on the efficiency of chromatographic step. One of the crucial chromatographic parameters is dynamic binding capacity (DBC) for certain biomolecule. DBC could be tailored with changing the surface area of convective pores by tailoring the surface of pre-polymerized monoliths using graft or block polymerization of polymer brushes. Grafted CIM monoliths have already been prepared via Radical Polymerization (RP) and successfully characterized (1).
Recently, the implementation and optimization of Controlled Radical Polymerization (CRP) for grafting of large pore monoliths (average diameter 6 μm ) resulted in polymethacrylate-based ionic exchanger with at least 5 times higher DBC compared to non-grafted 6 μm monoliths, while preserving high permeability. The main goal of our study was to chromatographically characterize novel grafted ion-exchanging monoliths (CIM gDEAE and CIM gSO3) to see whether novel columns still retain flow independent chromatographic properties of non-grafted monoliths.
To ensure the desired chromatographic characteristics of the CIM® monolithic column at large scales, monolith microstructure morphology, pore size distribution, porosity and surface ligand density should be uniform. To demonstrate the uniformity of large chromatographic monoliths we have developed new testing procedures. By fabricating smaller columns (disks) from different random positions of larger monolith, non-cGMP compliant chromatographic testing can be applied on the same polymerization batch without affecting the cGMP compliance of large-scale chromatographic monolith. Each individual disk was thoroughly tested and the results were compared to the properties of the large monolith.
There are many cases, where a single protein needs to be purified from a complex sample. Such proteins manifest themselves as impurities, which can affect further analysis, either by causing specific equipment malfunction or lower yield in the products. In other cases the specific protein is our molecule of interest, for example in glycomics analysis. In both cases high specificity for proteins, reproducibility and reliability is necessary. We have developed a model immunoaffinity column and 96-well plate based on an anti-fibrinogen monoclonal antibody, covalently immobilized onto CIMac™ analytical chromatographic monolith.
There are many cases, where a single protein needs to be purified from a complex sample. Such proteins manifest themselves as impurities, which can affect further analysis, either by causing specific equipment malfunction or lower yield in the products. In other cases the specific protein is our molecule of interest, for example in glycomics analysis. In both cases high specificity for proteins, reproducibility and reliability is necessary. We have developed a model immunoaffinity column and 96-well plate based on an anti-fibrinogen monoclonal antibody, covalently immobilized onto CIMac™ HDZ analytical chromatographic monolith.
Methacrylate monoliths (CIM® monolithic columns) allow for very fast and efficient separations and exhibit very high binding capacities for extremely large bio-particles due to their large inner channel diameters and enhanced mass transfer characteristics.
Additionally, the ability to manufacture polymer monolithic materials ranging from analytical to large scale preparative/industrial columns has tremendous advantages. By ensuring the chromatographic properties are consistent over the whole size range, one can easily design and optimize a purification method on laboratory scale and transfer it to a production line with minimal to no additional modifications.
Until now the largest monolithic column had a volume of 8 L, which was large enough to serve the biopharmaceutics' market's needs. Now however, the capacity of that column is already at its upper limit.
By successfully employing the knowledge and experience from almost two decades of monolith production we have managed to overcome the size limitations and polymerize the largest convective chromatographic support made from one piece of material, a 40 L monolithic column.
Immunoaffinity columns using antibodies as ligands against mammalian membrane proteins could be used for different applications in protein expresion control and, if a standard available, for concentration determination. Additionally these columns are ideal for polishing step of Fc fusion proteins of mammalian receptors.
Most importantly such columns could extract a significant amount of a pure membrane mammalian protein suitable for structural analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against MULT-1 transmembrane and RAE-1 GPI anchored glycoproteins were developed as a part of Glycomet project with the main goal to analyze the antigen glycan parts.
Two different preactivated support were used: hydrazide (HDZ) and carboxy imidazole (CDI).
Surface hydrophobicity/hydrophilicity of chromatographic stationary phases is one of the important characteristics that influence the chromatographic column performance. On the one hand, the surface should be highly hydrophilic to avoid nonspecific adsorption of sample molecules; on the other hand, the hydrophobic surface is crutial to e.g. separate the molecule isoforms.Therefore, fast and easy characterization method to evaluate the surface „hydrophobic/hydrophilic character" could be valuable.
First stage in the development of this method and the objective of this study was to evaluate the hydrophobicity of test set of 1 mL CIM columns with different ligand chemistries and densities. This was achieved by separation of protein mixture under hydrophobic interaction chromatography (HIC) conditions. Proteins were used since monoliths are used mainly in downstream of large biomolecules.
Moreover, since poor recovery under HIC conditions was observed on some columns, the research was additionally expanded with reversed phase chromatography (RPC) to obtain extra information about even more hydrophobic surface properties of monolithic columns. Therefore, after HIC step the RPC step followed and additional elution of proteins was achieved.
Phosphoproteomics is a branch of proteomics that focuses on deriving a comprehensive view of the extent & dynamics of protein phosphorylation by way of identifying & characterizing proteins that contain a phosphate group as a posttranslational modification. One of the approaches for specific enrichment of phosphopeptides from complex samples is metal oxide affinity chromatography (MOAC), where the specific adsorption results from bridging bidentate bindings formed between the phosphate anions and the surface of a metal oxide, such as TiO2, ZrO2, Fe2O3, and Al2O3. In presented study, a rutile TiO2 nanoparticles were bound to a previously polymerised CIM hydroxy monoliths.
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.
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.
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
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.
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.
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.
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.