Introduction
Packed-bed chromatography is recognized as a principal step in biotechnology processes. In this context, chromatography typically includes columns packed with resin to purify biomolecules based on chemical and physical differences. Resins are available by various adsorption surface chemistry, such as ion-exchange, affinity interaction, hydrophobic, or reversed-phase (Trilinsky and Lenhoff, 2007, Przybycien et al., 2004, Lyer et al., 2011). Varying concentrations of biomolecules are fed into the column and convectively flow between porous resin particles. The majority of binding occurs as the molecules diffuse into the pores. Biomolecules diffuse from the bulk liquid and flow into the resin pores to bind to the surface. Especially for larger biomolecules and viruses, diffusion can be the rate-limiting step and the flow rate is adjusted to ensure adequate time for diffusion into the particles. The dependence of flow rate on the diffusion is a significant limitation to overcome for biomolecules to bind to resins in columns. Relying on diffusion-driven flow increases recovery liquid volume required for elution (Roper and Lightfoot, 1995). In addition, high pressure drops can occur due to media deformation (Ghosh, 2002), and increase as a result of fouling during operation (Orr et al., 2013). Both of these limitations contribute to longer processing times and low throughput. Due to flow patterns, these conventional columns are often difficult and complex to scale-up for biotechnology processes. Scale-up often requires careful, uniform packing, which is not always consistent and can result in improper scale-up of equipment (Trilinsky and Lenhoff, 2007, Przybycien et al., 2004, Lyer et al., 2011, Roper and Lightfoot, 1995, Ghosh, 2002).
Membrane chromatography is an emerging technology for its effective removal of host cell DNA and proteins etc. through downstream purification. Overall, membrane chromatography describes the use of synthetic micro- or macroporous membranes that contain functional groups attached to the surface of internal pores for separation of biomolecules (Ghosh, 2002, Orr et al., 2013). Biomolecules convectively travel through these pores to bind, demonstrating a much more rapid flow pattern than via diffusion in resin-based chromatography. Convective flow patterns cause a low-pressure drop, lesser volumes of liquid, quicker processing times, and 100-fold higher throughput and efficiency. Generally, dynamic binding capacities are independent of flow rate based on the short residence time for biomolecules to bind to the membrane (Przybycien et al., 2004, Ghosh, 2002, Orr et al., 2013, Charcosset, 2006). Membrane chromatography is ideal for isolation of viruses and large biomolecules, and some studies have assessed their binding patterns on membranes (Wickramasinghe et al., 2006). Savings in time consumed, buffer usage, labor, and equipment dramatically decrease the overall cost, making membrane chromatography much more cost-effective than traditional resin-based chromatography (Przybycien et al., 2004, Orr et al., 2013, Charcosset, 2006, Warner and Nochumson, 2003).
Most popular in industry are radial flow adsorbers, where a flat sheet membrane is spirally wound over a porous cylindrical core. Membrane area increases radially outward, as well as axially within the membrane housing. Liquid flows through pores from the outer membrane cavity, to the inner core and exits through the inner core. The pressure within a given cartridge has been shown via CFD analysis to be independent of axial height, suggesting inlet pressure for each pore would be approximately the same (Teepakorn et al., 2016). Axially, in the housing inlet, biomolecules are transported mostly by convection. Optimal cartridge geometry (Teepakorn et al., 2015), flow distribution in the inlet of the housing (Ghosh, 2002), improved and varied (ie mixed matrix) membrane surface chemistries (Avramescu et al., 2003, Oksanen and Bamford, 2012) remains a focus for manufacturers of these units (ie Sartorius and Pall) as this promotes high throughputs and productivities for a range of biopharmaceutical processes. The Sartobind® Q membrane adsorber used in this study was designed to mimic flow patterns in larger scale Q membrane systems (Zhou et al., 2006) and similarly has quaternary ammonium groups covalently attached to a flexible hydrogel that is stabilized by a reinforced cellulose matrix. Hydrogel-grafted membranes have been shown to yield higher virus recoveries when compared to directly grafted membranes (Nestola et al., 2014). The specific functionalization of the membrane allows for adsorption of negatively charged biomolecules such as viral products, but could also bind host-cell DNA.
The goal of this research was to better understand and mathematically model viral product binding during the load step of the membrane adsorber. There have been other models that also considered the effects of convective flow and diffusion on the binding of biological products and impurities, some looking at the interaction between virus and cells (Gilbert et al., 2007) and some that studied the effects complex flow patterns inside membrane pores on single species binding (Ghosh et al., 2014). A recent study used a mathematical model that considered convection and diffusion to optimize the binding and elution of a Virus Like Particle (VLP) to the Sartobind Q membrane by varying the pH and ionic strength of the buffers (Ladd Effio et al., 2016). The focus of our research was to evaluate how well a simple and unique model, that assumes a two-species Langmuir-like isotherm and similar hydrodynamics and binding across all pores, predicts the effects of host cell DNA on viral recovery and system pressure during the process load step.
Section snippets
General process overview
The process being evaluated produces a non-envelope virus with size of 90–100nm. Material initially begins in the bioreactor and then undergoes DNA precipitation to reduce the level of host-cell DNA with a detergent. The material then undergoes filtration to remove any remaining detergent and large impurities (i.e HCPs) to produce clarified material (CM). Impurities are further captured for removal using anion-exchange chromatography with Sartobind Q membranes. Anion Exchange (AEX) eluate is
Physical characterization of the membrane
The pore radius (r) was determined from SEM analysis of the membrane (Fig. 3).
Based on the flowrate through the membrane and the porosity of the membrane, which is 0.798 (Ladd Effio et al., 2016); the average liquid velocity through a given channel (ie the chromatographic velocity) was estimated to be 2cm/min when the flowrate was 7.5ml/min. The “effective” pore length (l) was estimated to be 15mm, based on use of the Hagen-Poiseulle Equation and experimental pressure measurements at the
Discussion
The range of Dynamic Binding Capacities (Fig. 6) determined experimentally in this research (2.3–12.9mg/ml membrane) are consistent with that reported for binding of Virus Like Particles (5.7mg/ml) by Hubbuch (Ladd Effio et al., 2016). These experimental results indicate an interesting linear relationship (Fig. 6) between the pressure rise during the load step for this membrane adsorber and DBC (at 10% breakthrough) of the virus, This linear relationship was noted to require a certain amount
Conclusions
To date, there are few evaluations that compare the performance membrane chromatography across scales. Both scales considered here used Sartorius Sartobind® Q anion exchange membranes and exhibited similar trends of constant pressure rise The effect of flowrate and feed concentrations of virus and DNA on pressure rise observed during the loading phase of a membrane chromatography operation was evaluated through small-scale experimentation. The experimental behavior was consistent with an
CRediT authorship contribution statement
Kelsey O’Donnell: Investigation, Methodology, Formal analysis. Soumya Krishnathu: Methodology, Supervision. Ravinder Bhatia: Funding acquisition, Project administration. Zuyi Huang: Formal analysis. William Kelly: Conceptualization, Methodology, Funding acquisition, Project administration, Formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to recognize and thank Janssen for funding this research, and for their collaboration.
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Separation of tripeptides in binary mixtures using ion-exchange membrane adsorber
2023, Separation and Purification Technology
The adsorption of three tripeptides in an ion-exchange membrane adsorber was analyzed in single and binary solutions, with the aim of evaluating the capability of the membrane adsorber to separate triglycine (GGG) from two other tripeptides: glycine-histidine-glycine (GHG) and glycine-tyrosine-glycine (GTG). The equilibrium adsorption of single peptide solutions followed the Langmuir isotherm and GTG showed the highest adsorption affinity. The dynamic adsorption was fitted with a generalized model, which was defined using dimensionless parameters and based on the continuity equation. In general, the calculated and experimental breakthrough curves were correlated with high agreement. It was found that the axial dispersion coefficient was independent of the peptide molecule and that it increased with flow rate. The competitive adsorption between peptides in binary solutions was analyzed using the extended and modified Langmuir equations. The adsorption equilibrium data were satisfactorily fitted with the modified Langmuir isotherm for GGG/GHG solutions, while the extended Langmuir isotherm was a better fit to the data for GGG/GTG solutions. The experimental breakthrough curves of the two peptide binary mixtures were simulated using the parameters calculated from the competitive isotherms and the parameters obtained from the breakthrough curves of the single peptide solutions. The separation of GGG from the GGG/GHG mixtures was possible. The GGG recovery was higher than 35% and the GGG molar fraction in the outlet stream was higher than 0.994.
Paradigm shift from conventional processes to advanced membrane adsorption-mediated inactivation processes towards holistic management of virus - A critical review
2022, Journal of Environmental Chemical Engineering
Citation Excerpt :
The technical maturity aligned with advanced features of membrane operations have been developed from the scientific concept and mechanisms involved in basic conventional processes like adsorption, filtration, chromatography etc. The concept of size-exclusion mechanism based on the comparative size of membrane pore and viruses in pressure-driven microfiltration and ultrafiltration processes was evolved from basic filtration mechanism whereas, adsorption-mediated advanced membrane separation techniques for viruses removal has resemblance with chromatography techniques, where specific functional groups present inside internal pores help in interaction, attachment and separation of biomolecules [164]. Polymeric membranes made by polysulfone, polyethersulfone, polyethylenemine, plolyacrylonitrile, poly (ethylene terephthalate), polypropylene, polyvinylidine fluoride materials and ceramic membranes are most commonly used in water disinfection techniques due to lower energy involvement in the process while ensuring higher flux [56,165].
Since the earliest days, occurrence of deadly outbreaks caused by prevalence of different pathogenic virus infections affected human community periodically and caused millions of deaths per year. In order to improve safety and quality of human life against virus infections, it is imperative to do rigorous evaluation of existing virus treatment or elimination processes that would help to understand their potential merits, limitations and further prompts identifying the scope for developing advanced future technologies towards viral elimination. Considering the objective, present review comprehensively analyze present scope, potential and limitations of individual physical, chemical and biological methods in the direction of holistic management (recovery, purification, detection, elimination and disinfection) of viruses from liquid, solid, gaseous and interfacial environments. Among various treatment approaches, membrane filtration technology has been recognized as most promising, cost effective and energy efficient tool to deal with virus elimination from various resources. Membrane-based methods facilitates wide ranges of virus removal and disinfection capacity, mediated by size-exclusion, electrostatic or hydrophobic interactions and entrapment mechanisms. Based on the present demand of developing a complete and fully mature virus disinfection technique, potential of electro-spun nanofibrous membranes in selective capture and inactivation of viruses are clearly evaluated in the present review. Furthermore, it was found out from comprehensive analysis of available membrane mediated virus elimination processes that membranes derived from different forms of abundantly available, renewable and natural polymers such as cellulose are quite effective to capture, separate and inactivate viruses through depth filtration, charge based filtration and adsorption-oriented techniques.
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FAQs
How does anion exchange chromatography work? ›
Anion exchange chromatography is a form of ion exchange chromatography (IEX), which is used to separate molecules based on their net surface charge. Anion exchange chromatography, more specifically, uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges.
What are the disadvantages of membrane chromatography? ›A drawback of membrane chromatography is its low chromatographic efficiency and low binding capacity for proteins. A solution to these problems is to use monolithic columns. A monolith can be regarded as a piece of very 'thick' membrane.
What is the technology of membrane chromatography? ›Membrane chromatography is a large-scale separation method for separating, purifying, and recovering proteins and enzymes, which is a comprehensive protein purification technology.
What is the conclusion of ion exchange chromatography? ›Conclusion. Ion exchange chromatography is key for the separation of many organic and inorganic compounds and elements from a sample matrix and is widely used in geological research.
What are the factors affecting anion exchange chromatography? ›The factors that affect separation during ion exchange chromatography include the surface area of the stationary phase (resin bead size); the density of exchange sites on the stationary phase surface (cross-linkage); the flow rate of the mobile phase (resin bead size and column geometry; system pressure in high- ...
What are the limitations in chromatography experiment? ›Techniques for paper chromatography cannot distinguish between volatile compounds. Paper chromatography is not able to handle large sample volumes. Quantitative analysis could be more helpful in paper chromatography. Paper chromatography cannot separate complex mixtures.
What is the problem with chromatography? ›There is a problem which arises in all types of chromatography, when samples of widely differing retention properties are present in the same sample. If the elution conditions are correct for the early eluting compounds, the late ones will remain in the column too long.
What is everything about ion exchange chromatography? ›Ion-exchange chromatography separates molecules based on their respective charged groups. Ion-exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. The ion exchange chromatography matrix consists of positively and negatively charged ions.
What are the two types of ion exchange chromatography? ›Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exchange and anion exchange chromatography.
What can the chromatography technique be successfully used for? ›Chromatography is a separation technique used to separate the different components in a mixture. The purpose of preparative chromatography is to separate the components of a mixture for more advanced users and thus a form of purification.
What are the advantages and disadvantages of ion exchange chromatography? ›
Advantages and disadvantages of Ion exchange chromatography
The separation can be altered by changing pH of the buffer, salt gradient, or by changing the nature of ion exchange resin. Separation time is short hence large sample volumes can be separated in a short period of time. It has a low maintenance cost.
Ion-exchange chromatography (IEX) depends on the interaction of charge on the surface of a protein with an opposite charge on an insoluble matrix.
What are the advantages of anion exchange chromatography? ›Anion-exchange chromatography with pulsed amperometric detection has long been the main method for the analysis of monosaccharides [133, 134]. The main advantage of this technique is its ability to analyze monosaccharides without derivatization, and to provide good resolution.
What will elute first in anion exchange chromatography? ›A compound with a lower charge will elute first. The stationary phase would have charges opposite those of the ions that we wished to separate. For example, maybe the column is packed with a phase that contains many anions.
What is the common buffer for anion exchange chromatography? ›| Substance | pKa | Working pH |
|---|---|---|
| Piperazine | 5.68 | 5.2-6.2 |
| Bis-Tris | 6.5 | 6.0-7.0 |
| Bis-Tris propane | 6.8 | 6.3-7.3 |
| Triethanolamine | 7.8 | 7.25-8.25 |
The dilution times of the samples and the dilution tools are the sources of errors. As the main force of injection, it is still the manual injector. If used incorrectly, it can cause chromatographic problems, lack of linearity in standard curves, and poor repeatability.
What is the biggest hazard when performing a chromatography experiment? ›Uncontrolled release of toxic material is one of the most dangerous chromatography hazards both for users and for their surroundings. There, chemists could invest in chromatography equipment that can offer them the best protection possible.
What are some possible sources of error in the column chromatography experiment? ›Possible sources of error include: Failure to properly measure the volume of liquid in containers. (Rendering further calculations inaccurate.) Failure to apply steady pressure.
What can limit the effectiveness of chromatography? ›Rf values and reproducibility can be affected by a number of different factors such as layer thickness, moisture on the TLC plate, vessel saturation, temperature, depth of mobile phase, nature of the TLC plate, sample size, and solvent parameters.
What are the disadvantages of chromatographic data analysis? ›The main disadvantage of these powerful analytical platforms is that they are restricted to a laboratory environment due to the lack of portability features. In addition, multi-stage sample preparation requires time-consuming analysis, and the maintenance costs are relatively high [17] . ...
What is the disadvantage of ion chromatography? ›
One of the main disadvantages of ion exchange chromatography is its buffer requirement: because binding to IEX resins is dependent on electrostatic interactions between proteins of interest and the stationary phase, IEX columns must be loaded in low-salt buffers.
How effective is chromatography? ›Chromatography methods based on partition are very effective on separation, and identification of small molecules as amino acids, carbohydrates, and fatty acids. However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins.
How can I improve my chromatography results? ›- Increasing column length.
- Decreasing particle size.
- Reducing peak tailing.
- Increasing temperature.
- Reducing system extra-column volume.
The Rf value of a component in Chromatography can be affected by several factors, including the type of stationary phase, the polarity of the solvent, the temperature, and the concentration of the components in the mixture.
What does anion resin remove? ›Cation and anion resins remove dissolved ionic contaminants.
What is the process of anion exchange? ›Anion exchange is the process in which anions in the form of acids are adsorbed by a basic substance. It describes the exchange of ions in which one anion (as chloride or hydroxide) is substituted for one or more other anions (as sulfate). It is highly effective on negatively charged ions such as: Bicarbonate.
What are the three application of ion exchange chromatography? ›Applications of ion-exchange chromatography include the analysis of a wide range of organic and inorganic ions. Biological applications include the analysis of amino acids, proteins or peptides, and polynucleotides [7]. Fekete et al.
What is an example of a real life application for an ion exchange resin? ›Ion exchange resins find a number of applications in fruits and beverage industry for improving taste and flavour through removal of undesirable components. Common applications include removal of trace metals, bad taste and smell, decolouration and primary treatment of water used in manufacture of juices and drinks.
What is the example of ion exchange chromatography? ›Example buffers used for ion-exchange chromatography include: Tris, phosphate, acetic acid, and triethanolamine. 2.7. Schematic of the ion-exchange process for an anion-exchange resin: (a) negatively charged protein displaces the chlorine anions due to its greater negative charge at the pH of the mobile phase.
What binds to an anion exchange column? ›Negatively charged glycans such as sialic acids bind to the anion exchanger, while neutral sugars pass through the column.
What are 2 examples of chromatography being used for in real life and why is it important? ›
The Police, F.B.I., and other detectives use chromatography when trying to solve a crime. It is also used to determine the presence of cocaine in urine, alcohol in blood, PCB's in fish, and lead in water. Chromatography is used by many different people in many different ways.
What are two reasons why chromatography is useful in scientific research? ›Chromatography is a method that is used in laboratories for the separation of a mixture. It is used to test drug levels and water purity. It is also used to determine the nutritional value of the food sample. It is used to determine the type of chlorophyll in various photosynthetic organisms.
What are the steps of anion exchange chromatography? ›The basic process of chromatography using ion exchange can be represented in 5 steps: eluent loading, sample injection, separation of sample, elution of analyte A-, and elution of analyte B-, shown and explained below. Elution is the process where the ion of interest is moved through the column.
How does separation occur in anion ion chromatography? ›Ions will move through the columns of the ion chromatographer at different speeds depending on their affinity for the specific resin, and they will separate from each other based upon differences in ion charge and size.
What is a simple explanation of ion exchange chromatography? ›Ion exchange chromatography (IEX) is a chromatographic separation method essentially based on the net charge of the protein. Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins.
What are the six 6 steps in regenerating a bed of anion or cation resin? ›- Backwash. During the service cycle, the resin bed collects suspended impurities from water. ...
- Regenerant Introduction. Regenerants of proper concentration are introduced into the tanks to reactivate the resin. ...
- Displacement (Slow) Rinse. ...
- Fast Rinse.
Proteins with weak ionic interactions are the first to elute from the column. In the case of anion exch- ange chromatography, proteins that are less negativly charged start to elute first. With an increase of the salt concentration proteins with stronger ionic interaction elute later from the column.
What is the purpose of anion exchange? ›Anion exchange chromatography is commonly used to purify proteins, amino acids, sugars/carbohydrates and other acidic substances with a negative charge at higher pH levels. The tightness of the binding between the substance and the resin is based on the strength of the negative charge of the substance.
What does anion exchange remove? ›What is anion exchange? With anion exchange, water passes through a resin bed which removes arsenic by exchanging it for a non-toxic substance attached to the bed. Once the space on the resin bed is full, the system backwashes with brine to regenerate the bed.
What does an anion exchange membrane do? ›Anion exchange membranes (AEM) are membranes which have positively charged functional groups. These membranes allow the transport of anions, while rejecting cations.
What are the advantages of ion exchange chromatography? ›
Advantages of ion exchange chromatography
1. t is one of the most efficient methods for the separation of charged particles. 2. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Size, size distribution and porosity of the matrix particles are the main factors which affect the flow characteristics and chromatographic resolution.
What are the drawbacks of ion exchange? ›Disadvantages of Ion Exchange
Long-term costs are high when it comes to operating ion exchange equipment. Unable to effectively remove bacteria from water. While ion exchange beds can be regenerated, salt water is sent directly into the environment during this process.
Uses for ion exchange chromatography
separation of proteins from foods, for example, to investigate the effects of individual food components on health – this type of analysis is used in nutrition research. separation of high value proteins from substances. drinking water analysis for pollution and other constituents.