Literature Review on the Ion Exchange and Size Exclusion Chromatography Kevin Tan Jia Wei Khoo Yong Jie Ang Jun Wei CHEM-E3140 Bioprocess Technology II Instructor: Sandip Bankar Date of Submission: 5 December 2017 Declaration By completing this cover sheet and declaration, I confirm that this assignment is my own work, is not copied from the work (published or unpublished) of any other person, and has not previously been submitted for assessment either at Aalto University, or another educational establishment. Any direct or indirect uses of material (e.g.: text, visuals, ideas ) from other sources have been fully acknowledged and cited according to the conventions of the Harvard Referencing System.
Contents 1. Abstract... 3 2. Background... 4 Chromatography... 4 3. Mechanism... 5 Ion Exchange Chromatography (IEC)... 5 Size-Exclusion Chromatography (SEC)... 6 4. Disadvantages of IEC and SEC setups... 8 5. Importance within the bioprocess industry... 9 Ion-Exchange Chromatography (IEC)... 9 Size-Exclusion Chromatography (SEC)... 9 6. Conclusion... 10 7. References... 11
1. Abstract In the bioprocess industries, reactors involving microbes are used for many purposes, such as the synthesis of specific proteins for drug manufacturing. However, due to unwanted side reactions and waste products produced by the microbes, the downstream product usually consists of the desired protein immersed in numerous impurities. In the case of drug manufacturing, it is especially important that the final product is pure. This article provides insight on the use of chromatography, specifically Ion-Exchange chromatography and Size- Exclusion Chromatography for separation. This report serves to deepen our understanding on the mechanisms behind these chromatography techniques and also its specific shortcomings. Moreover, it also serves to emphasize on the importance of these techniques by analysing some of its crucial applications within the bioprocess industries.
2. Background Since the usage of the fermentation in food preservation and production of beer and wine since ancient times, fermentation processes have gone a long way and are used in the production of most biological products now. Developments in product separation have been an important part in the development of these processes, as most fermentation processes do not go to completion and hence many unwanted by-products are present together with the product in concern. Product separation refers to the segregation of materials after its production process. Depending on the product, different components of the final mixture have to be separated and purified to retrieve the pure final product. Chromatography Chromatography is commonly used in biotechnology for purifying biological molecules, like proteins, for medicine or other uses. Chromatography allows the separation of individual components from complex mixtures. It consists of a mobile phase, which consists of the solvent and product mixture, and a stationary phase through which the mobile phase travels. Some examples of the stationary phase include paper, and glass beads, commonly known as resins. Figure 1: Chromatography setup Figure 1 above shows the typical setup of a lab chromatography column. The mobile phase is added to the stationary column and gravity pulls the mobile phase downwards, causing the proteins present in the mixture to separate and elude from the columns in separate and distinct portions. Molecules travel through the stationary phase at different rates because of their unique properties, such as weight, size or electronic charges. Different chromatography setups utilizes the differences in one of these properties to separate mixtures of different compounds. Some examples include: Ion Exchange Chromatography (IEC), which separates compounds with different charges using charged resins as the stationary phase.
Gel filtration chromatography, commonly referred to as size exclusion chromatography (SEC), which separates compounds with different molecular sizes. In SEC, the stationary phase consists of resins which contain tiny holes, which allows some smaller molecules to pass through these pores. Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, or receptor and ligand. It is able to target a specific compound for separation, and the stationary phase would contain the complementary molecule. This separation technique has the highest molecular specificity. In this article, we will be focusing on IEC and SEC. 3. Mechanism Ion Exchange Chromatography (IEC) Ion Exchange Chromatography (IEC) utilizes the principle of the electrostatic forces of attraction to separate a mixture of charged proteins. Charged proteins are classified into positively and negatively charged proteins based on the presence of different charged functional groups. At a ph of 7, amino acid groups such as Histidine, Arginine and Lysine are positively charged while Aspartic acid and Glutamic acid are negatively charged. Figure 2: Charged amino acid groups The apparatus involved in Ion Exchange Chromatography (IEC) consists of a chromatography column filled with beads of a particular charge (stationary phase), opposite to that of the protein of interest. When the protein mixture (mobile phase) is poured through the chromatography column, proteins with the same charge as the charged polymer beads are repelled and eluded from the column rapidly. These proteins are then collected in a flask at the bottom of the column as the effluent. On the other hand, proteins with a complementary charge are attracted to the charged polymer beads and remain in the column as the residue.
Figure 3: Mechanism of Ion Exchange Chromatography (IEC) Figure 3 shows the mechanism for a cation exchange system, with the anion exchange using positively charged resins instead to bind to the negatively charged proteins. After removal of the effluent, a salt buffer solution such as NaCl solution is utilized as an eluding agent. When poured down the chromatography column, the charged ions within the buffer solution would compete with the protein molecules for binding to the polymer beads. As a result, the residue will then be eluded and collected. When separating proteins with differing magnitude of charges, buffer solutions of increasing concentrations can be used to separate these proteins. Size-Exclusion Chromatography (SEC) Size-Exchange Chromatography (SEC) utilizes the principle of varying protein molecular sizes for separation. The apparatus required involves a porous polymeric material with numerous microscopic holes being placed within the chromatography column. The sizes of these holes are determined by the sizes of the proteins, as it must allow some molecules to pass through while preventing others from doing so. The imperfect packing of the resin means that there are gaps between the resin particles, and also with the column walls where the larger molecules will move through. Figure 4: Mechanism of Size-Exclusion Chromatography (SEC) The protein mixture is first poured into the column and protein molecules larger than the pores of the polymer beads (shown in red) are unable to fit through. Instead, they flow
downwards rapidly and are collected as eluent. For proteins with a molecular size smaller than the polymeric pores (as shown in blue and green in Figure 4), they flow through the polymer at a slower rate and are only collected after all the bigger protein molecules have been eluded. The eluded protein mixture would then be poured into another chromatography column consisting of polymeric beads with a smaller pore size than the first column. The protein mixture that flows through the column rapidly can be concluded to have a molecular size ranging from the pore size of the first to the second polymeric material. As explained above, the smaller protein molecules would be entrapped within the pores of the polymeric beads and spend a longer time within the column before being eluded. Numerous repetitions involving chromatography columns of decreasing polymeric pore sizes would be required in order to obtain an efficient separation of the original protein mixture.
4. Disadvantages of IEC and SEC setups One of the disadvantages present in chromatography setups is that it is not specific in the types of molecules being filtered. Even though repeated iterations of IEC and SEC will be able to remove many of the impurities present, on its own, it is unable to perfectly purify a substance, due to the presence of either other impurities of similar ionic charges or molecular sizes respectively. Another disadvantage is that these systems are less efficient than other methods. Firstly, for molecules that are similar in affinity to the static phase, it takes a very long column bed to ensure there would be sufficient separation between the 2 molecules so that they can be separated completely. This would also mean that it takes a very long time for their separation, and also that an increasing portion of the column becomes redundant as the compounds are already separated, as can be seen in figure 5, where the bottom of the first column and top of the last column are unutilised. Figure 5: Chromatography process Moreover, the chromatography process cannot happen continuously as a fixed length of the column will only be able to separate a certain amount of mixture effectively, which means that the system can only be scaled up linearly, through the repeated use of the system instead of separating a larger quantity at once. In the laboratory scale, these disadvantages can be solved by using a moving bed chromatography system, where the static and mobile phase are moved in opposite directions. Solvent à Stationary Phase ß à Solvent ß Stationary Phase Slower Feed Faster Molecule Molecule Figure 6: Moving bed chromatography As see in figure 6, if the relative speeds of the resins and solvent are adjusted accordingly, the faster molecule will be collected to one side of the feed while the slower one collected on the other side.
In the industrial scale however, it is a complex process to move the 2 phases and hence a simulated moving bed configuration is used instead, whereby the inputs (feed and solvent), and outputs (extract and raffinate) are moved instead. This system is also known as the simulated moving bed chromatography and can be used to scale up all chromatography separation techniques. This system is used widely in the biological and pharmaceutical industries to separate products instead of the conventional column chromatography. 5. Importance within the bioprocess industry Ion-Exchange Chromatography (IEC) Glucose-Fructose separation IEC is used to separate fructose from glucose from corn. After the breaking down of the starch by amylases, the composition of the fermentation mixture then comprises of 50% glucose, 42% fructose and unconverted oligosaccharides. Fructose is then purified to 90% purity using a Ca based ion-exchange chromatography before being either sold or mixed with the original sugar solution to form a 55% fructose - 45% glucose mixture, also known as high fructose corn syrup(hfcs). The production of HFCS has been a breakthrough in the food industry, especially when many countries are passing sugar taxes, which makes the originally cheaper beet sugar a more expensive sweetener. Size-Exclusion Chromatography (SEC) Desalting In bioprocess reactors, batches of pure protein molecules have to be obtained in order to pass the stringent requirements stipulated by pharmaceutical companies. As such, essential salts and ions introduced into the reactors to promote microbe growth have to be removed from the resultant protein solution in a purification process known as desalting. Desalting utilizes the principle of size-exclusion chromatography to separate the smaller salt molecules from the larger protein molecules of interest.
Figure 7: Mechanism of Desalting From Figure 7 above, the smaller salt molecules and ions which are able to fit within the pores of the resin beads are entrenched within the beads, taking a longer time to be eluded. On the other hand, due to its larger molecular size, the protein of interest flows around the resin beads and are eluded instantaneously. As such, a pure sample of protein can be obtained. An example of an industrial application involves the separation of the protein ubiquitin in pharmaceutical industries from impurities such as sodium chloride (NaCl) and Vitamin B12 present within the fermentation broth by utilizing desalting columns as shown in Figure 8 below. 6. Conclusion Figure 8: A spin desalting column All in all, chromatography techniques such as ion-exchange chromatography and size exclusion chromatography play essential roles in the purification of downstream products within the bioprocess and pharmaceutical industries. However, these techniques do possess its unique shortfalls and cannot be applied universally to every situation. More often than not, a mixture of chromatography techniques has to be applied in order to obtain pure samples of the desired protein from the initial feed protein mixture. In order to fully utilize these chromatography techniques, it is crucial to first understand its mechanism and the nature of the compounds present in the mixture before deciding on the suitable separation process.
7. References Custom Size-Exclusion Chromatography Service. (2017). Creative-biostructure.com. Retrieved from https://www.creative-biostructure.com/custom-size-exclusionchromatography-service-259.htm Desalting and Gel Filtration Chromatography. (2017). Thermofisher.com. Retrieved from https://www.thermofisher.com/fi/en/home/life-science/protein-biology/protein-biologylearning-center/protein-biology-resource-library/pierce-protein-methods/desalting-gelfiltration-chromatography.html Ion Exchange Chromatography (IEX) Biopharmaceutical Manufacturing Merck. (2017). Merckmillipore.com. Retrieved 5 December 2017, from http://www.merckmillipore.com/fi/en/products/biopharmaceuticalmanufacturing/downstream-processing/chromatography/ion-exchangechromatography/joab.qb.tkgaaafab.zkiqpx,nav Amino acids - Dynamical Systems @ CFisUC. Retrieved from http://condmat.lca.uc.pt/?page_id=1007 Coskun, O. (2017). Separation Tecniques: CHROMATOGRAPHY. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5206469/ Viard, V., & Lameloise, M. (1992). Modelling glucose-fructose separation by adsorption chromatography on ion exchange resins. Journal Of Food Engineering, 17(1), 29-48. http://dx.doi.org/10.1016/0260-8774(92)90063-c Al Eid, S. (2006). Chromatographic separation of fructose from date syrup. International Journal Of Food Sciences And Nutrition, 57(1-2), 83-96. http://dx.doi.org/10.1080/09637480600658286 Zeba Desalting Products Retrieved from https://www.thermofisher.com/fi/en/home/lifescience/protein-biology/protein-purification-isolation/protein-dialysis-desaltingconcentration/zeba-desalting-products.html