1. NOVEL METHODS IN BIOSEPARATION (Downstreamprocessing recentissues) By: Naghmeh Poorinmohammad May 2015 2. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 2 3. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 3 4. Introduction Increasing number of rDNA Products Progress in Cell culture Demands for higher product yields Need for novel approaches for fast and cost-effective DSP 4 5. Most of the increases in productivity of biomanufacturing achieved in previous decades have resulted from improvements in the upstream production phase. Downstream processing is now routinely found to be the bottleneck in because its capacity has not kept pace with upstream production . Introduction General solutions Streamlining of existing processes Use of innovative technologies 5 6. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 7 7. Mix&GoTM Glue Protein purification using magnetic particles is well established in life science R&D but has not reached commercial acceptance for larger scale applications in bioprocessing. Every magnetic particle being used requires the coating of iron oxide nanoparticles (magnetite) with a polymer casing to achieve various desirable characteristics including the ability to efficiently bind protein or other ligands onto its surface. The manufacturing process often requires multiple steps and achieving reproducibility and consistency of performance from large scale production batches is not trivial when dealing with micron and sub-micron particles. Consequently, magnetic particles retail in the order of $1000 per gram, with some suppliers being dramatically more expensive. 8 8. Mix&GoTM Glue Anteo avoids the need for multiple coatings by directly coating the magnetite with a metal polymer-based surface chemistry called Mix&GoTM (patented in 2013). The procedure is simple and consists of adding sonicated magnetite to a Mix&Go solution and stirring for an hour. Scale up issues should be trivial even at multi-kg and higher scales. 9 Adopted from: http://anteotech.com/produ ct/smpn100/ 9. It uses metal ions in polymeric form in an aqueous solution to attach proteins to synthetic surfaces via chelation and coordination chemistry. It relies on avidity binding to hold proteins in place strongly. Polymeric metal ions form coordinate bonds with electron donating groups on synthetic surfaces on the one side and biomolecules on the other. A single chelation point is not strong enough to bind a metal ion to a synthetic surface or to bind a biomolecule to the metal ion. Anteos platform technology overcomes this limitation by using polymeric metal ions that form multiple chelation points with both the underlying surface and the biomolecule. Together many such interactions form a very strong, yet flexible bond that is as secure as a covalent bond. Mix&GoTM Glue 10 10. Mix&GoTM Glue 11 Adopted from: http://anteotech.com/technology/characteristics/ 11. Protein A was coupled to the Mix&Go activated particles and to Mix&Go activated Life Technologies M270 magnetic particles, and their antibody loading capacities were subsequently compared to commercially available Protein A Life Technologies M270 particles. Mix&GoTM Glue Vukovic, P. "Simple And Versatile Protein Immobilisation Onto Surfaces By Multi-Point Non-Covalent Metal Chelation." 12. Mix&GoTM Glue Other advantages of Mix&Go Resistant to damage Fast, Stable and Reproducible Broad applicability 13 13. Mix&GoTM Glue 14 Adopted from: http://anteotech.com/technology/characteristics/ 14. Mix&GoTM Glue 15 Adopted from: http://anteotech.com/technology/c haracteristics/ 15. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 16 16. The major interactions involved in the protein adsorption onto polymeric membranes mainly include hydrophobic interaction, electrostatic interaction and hydrogen bonding , in which hydrophobic interaction is the most important one during adsorption process. Therefore, hydrophobic hydrophilic transition characteristics of polymeric materials can be used as a key to open the possibility of modulating the protein adsorption and release behaviors. PNIPAM as a partner of PMMA to fabricate a thermo-responsive nanofibrous membrane for recycling application in separation of biomolecules. PNIPAM is a kind of responsive polymer, which is hydrophobic above the LCST and hydrophilic below LCST. Nanofibers based on copolymers containing PNIPAM were reported to possess thermo-responsive properties. A Novel thermo-responsive nanofibrous mat 17Song et al. European Polymer Journal 47.10 (2011): 1885-1892. 17. Swellingdeswelling behavior of the blend nanofibrous mat was verified to be thermo-responsive. Moreover, the PMMA/PNIPAM blend nanofibrous mats showed reversible swelling deswelling behavior, and the property can be used to adsorb and release protein repeatedly. A Novel thermo-responsive nanofibrous mat 18Song et al. European Polymer Journal 47.10 (2011): 1885-1892. 18. A Novel thermo-responsive nanofibrous mat 19Song et al. European Polymer Journal 47.10 (2011): 1885-1892. 19. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 20 20. A variety of proteins, domains, and peptides have been used as affinity tags to facilitate the purification of proteins of interest from crude extracts. The purification of a target protein using an affinity handle offers several advantages over the conventional chromatographic methodologies. What are they? An affinity tag is often chosen taking into account the purification costs: different affinity media and elution principles present different expenses during the operation process and should therefore be carefully selected at the beginning of the cloning strategy. Fusion tags for affinity purification 21 21. Fusion tags for affinity purification 22Costa et al. Frontiers in microbiology 5 (2014). 22. The novel Fh8 fusion system (hitag) Fh8 is a small secreted antigen by F. hepatica. Recombinant Fh8 produced in E.coli led to the development of immunodetection of the infection. When produced recombinantly it was understood that Fh8 is highly soluble in E-coli and is thermostable. 23Costa et al. Frontiers in microbiology 5 (2014). 23. The novel Fh8 fusion system (hitag) Fh8 is a Ca2+ sensor protein that opens its structure upon calcium accommodation. The opening of the Fh8s structure exposes a large hydrophobic surface that becomes available for interaction with its targets The Fh8 tag and Fh8-fused proteins presented a calcium-dependent interaction with a hydrophobic resin, and, as reported for other calcium-binding proteins this interaction was still occurring even with low salt concentration in the mobile phase. Moreover, it was also shown that, as a calcium-binding protein, the Fh8 tag and Fh8- fused proteins can be eluted by using a calcium chelating agent, such as EDTA. This elution strategy allows a single-step and rapid elution of all bound proteins 24Costa et al. Frontiers in microbiology 5 (2014). 24. 25Costa et al. Frontiers in microbiology 5 (2014). 25. The novel Fh8 fusion system (hitag) Fh8 r-PPure System The Fh8 is ranked among the best solubility enhancer tags as Trx, MBP, or NusA and it offers a specific and simple purification of the target proteins by using its natural calcium-binding properties and mild conditions for HIC. It is one of the few existing tags to promote simultaneously target protein solubility directly into the E. coli cytoplasm and a simple and cost-effective protein purification. 26 26. Another novel Fusion tag B. cereus spore coat protein CotB1 and its C-terminal 14-aa peptide CotB1p bind to silica surfaces. The 14-aa CotB1p region of CotB1 plays a crucial role in binding to silica. The amino acid sequence of CotB1p (SGRARAQRQSSRGR) is rich in positively charged arginine residues. Therefore has a high net positive charge. In contrast, the surface of silica is negatively charged under either neutral or basic conditions electrostatic attraction is a major driving force behind the binding of CotB1p (and also CotB1) to silica. The resulting fusion proteins can be easily immobilized on silica surfaces by mixing the proteins in a solution with silica materials. 27Abdelhamid et al. Applied microbiology and biotechnology 98.12 (2014): 5677-5684. 27. Another novel Fusion tag Abdelhamid et al. Applied microbiology and biotechnology 98.12 (2014): 5677-5684. 28. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 29 29. 1-vinyl-3-butylimidazolium chloride (ViBuIm+Cl)IL monomer N,N 0-methylenebisacrylamide (MBA) cross-linker. Ionic liquid polymer 30Yuan et al. Journal of Materials Chemistry 22.9 (2012): 3965-3972. 30. 31 Ionic liquid polymer Yuan et al. Journal of Materials Chemistry 22.9 (2012): 3965-3972. 31. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 32 32. Monolithic Cryogels 33Kumar and Srivastava. Nature protocols 5.11 (2010): 1737-1747. 33. 34 Monolithic Cryogels Ertrk and Mattiasson Journal of Chromatography A 1357 (2014): 24-35. 34. 35 Monolithic Cryogels Kumar and Srivastava. Nature protocols 5.11 (2010): 1737-1747. 35. 36 Monolithic Cryogels Ertrk and Mattiasson Journal of Chromatography A 1357 (2014): 24-35. 36. 37 Monolithic Cryogels Ertrk and Mattiasson Journal of Chromatography A 1357 (2014): 24-35. 37. 38 Monolithic Cryogels Ertrk and Mattiasson Journal of Chromatography A 1357 (2014): 24-35. 38. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 40 39. Core-shell particles 41 Solid-core particles coated with a thin layer of stationary phase. Got their start in the 1960s. Commrecial versions began appearing in their in 2007. limited solute penetration depth in the shell, thereby improving mass transfer. Require one-half to one-third the pressure to operate than do columns of the same overall dimensions packed with totally porous particles *. PRACTICAL EXAMPLE: DeGrasse et al. separated shellfish toxins and reported that using a core-shell stationary phase compared to fully porous particles saved two-thirds of the analysis time**. * Chester, Analytical chemistry 85.2 (2012): 579-589. ** DeGrasse et al. Toxicon (2011): 57, 179-182. 40. 42 Core-shell particles Chester, Analytical chemistry 85.2 (2012): 579-589 41. Molecularly Imprinted Polymers (MIPs) Turiel and Martin-Esteban Analytical and bioanalytical chemistry 378.8 (2004): 1876-1886. 42. Molecularly Imprinted Polymers (MIPs) 44Turiel and Martin-Esteban Analytical and bioanalytical chemistry 378.8 (2004): 1876-1886. 43. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 45 44. Single-use disposable technologies Benefits Elimination of cleaning requirements Reduced risk of cross-contamination Faster turnaround between campaigns Reduced time for a new facility to become operational Increased convenience and flexibility 46 45. Many different factors have combined to encourage the current surge of interest in single-use or disposable technologies for biopharmaceutical manufacturing Single-use disposable technologies Shukla et al. Trends in biotechnology 31.3 (2013): 147-154. 46. Single-use disposable technologies 54Langer et al. Engineering in Life Sciences 14.3 (2014): 238-243. 47. Single-use disposable technologies 55Langer et al. Engineering in Life Sciences 14.3 (2014): 238-243. 48. Unit operation Examples of single-use technology on the market for DSP Centrifugation - kSep1 400 and 6000 (for 16000 liters) - Carr Unifuge for up to 1000 liters Chromatography columns - ReadyToProcess (GE) - Opus (Repligen) - GoPure (Life Technologies) - Uno monolith (BioRad) - CIM1 monolithic columns (BIA Separations) Depth filtration - POD (Millipore) - Stax (Pall) - Zeta Plus (Cuno) - Sartoclear P (Sartorius-Stedim) Membrane chromatography - Mustang (Pall) - Sartobind (Sartorius) - Chromasorb (Millipore) Viral filtration - Planova 15N and 20N (Asahi) - Viresolve Vpro (Millipore) - Virosart CPV (Sartorius) - DV20 (Pall) 56 49. Centrifugation has proved to be more difficult to convert into a single- use setup because of the complexity of the devices compared to filters. However, two disposable centrifuges are now commercially available: 1. KSep Systems has a technology based on revolving chambers that are fitted with a single-use bag to enable closed system processing. 2. Carr Centrifuges has launched the Unifuge system that is essentially a tubular bowl centrifuge lined with a bag. SUS: Centrifugation 57 50. kSep Systems patented kSeptechnology is the only current technology that provides significant advantages for users that want to either harvest cells as product or discard cells as by-product during manufacturing. It is a Closed system with single-use Class VI product contact surfaces. 58 51. 59 52. Feed In Concentrate Out Supernatant Out Ultrasonic sensor Peristaltic feed & harvest pump RPM Sensor Cooling jacket supply connect Onboard air supply and sterile filter Fiber optic level sensor Single-use pressure sensor 60 53. Rigid + flexible components Tube set is integrated as part of single use component Terminally sterilized (gamma) Complies with: ISO 10993 USP & Class VI plastics Polycarbonate Polyurethane Polypropylene C-Flex Silicone 61 54. 62 55. Cell Line Volume Flow rate G-force Feed Viability Harvest Viability % Loss in supernatant CHO 56L 3 L/min 2,000 99% ~98% 1.68% CHO 50L 4 L/min 3,000 95.4% 97.3% Negligible CHO 57L 5 L/min 3,000 98.6% 98% 2.97% CHO 90L 4 L/min 3,000 96.2% 98.6% - 63 56. Unit(s) available for field testing in US and Europe Typical field trial period is for one (1) week and includes on site technical support Each customer will purchase the disposable liner(s) needed for testing The beta test single-use components will not be gamma irradiated Unifuge Pilot is available for sale, estimated delivery is 12 weeks 65 57. 66 58. The system operates with ready-to-use, disposable flow paths that are available separately in High and Low Flow Kits. As a benefit, the need for cleaning between products/batches is eliminated and no development and validation of cleaning procedures is required. Replacing flow paths between batches is fast, and when used together with ReadyToProcess columns, the risk for cross-contamination is removed. SUS: Chromatography column 67 59. SUS: Chromatography column 68 60. GE Healthcare Life Sciences ReadyToProcess normal flow filter capsule assemblies are disposable normal flow filters designed for laboratory through process scale processing, with typical volumes ranging from a few hundred milliliters to 1000 L of solution. SUS: filteration 69 61. Another challenge that affects all disruptive technologies is the absence of standardization and regulation of the quality of materials used. The plastic bags used for cell culture have the potential to either bind media components or to contribute leachables that could adversely impact cell growth. scalability is one of the current challenges for vendors of disposable technologies, future commercial production will likely be carried out at the 5000 liter scale because of the increasing titers of cell cultures. disposing plastics? SUS drawbacks 71Langer et al. Engineering in Life Sciences 14.3 (2014): 238-243. 62. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 72 63. Stirred well filtration(SWF) There is considerable interest in the use of microscale processing (MSP) techniques for the development and optimization of biological manufacturing processes. WHY? 1. Reduction of costs 2. Reduction of time 3. Can be automated using robotic handling systems SWF: 96-well filter plates with a magnetic tumble stirrer that simultaneously mixes the solution above the membrane in each well. SWF: A high-throughput technique for downstream processing studies The technology offers high throughput experiments to be used in DOE. 73Kazemi et al. Journal of Membrane Science 470 (2014): 30-39. 64. Stirred well filtration(SWF) 74Kazemi et al. Journal of Membrane Science 470 (2014): 30-39. 65. In order to demonstrate the usefulness of the SWF technique for filtration process optimization, a set of protein solutions were prepared from different buffer solutions and then tested simultaneously in the multiple wells of the filter plate. Stirred well filtration(SWF) solution pH Solution ionic strength 75Kazemi et al. Journal of Membrane Science 470 (2014): 30-39. 66. What are the other possible factors to be chosen for filtration process optimization using SWF? Stirred well filtration(SWF) Membrane modification Filtrate flux Stirring effect Membrane type 76 Membrane fouling 67. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 77 68. Simulatedmoving bed chromatography (SMBC) 78 The theory is not new! It was proposed in 1950s! But why it is put here? Simulated moving bed chromatography was searched on Google Scholar: 1950-2000 23800 results 2000-2015 89200 results In recent years, the method is vastly being used and investigated practically, while most of papers of 1950-2000 were based on the concept of the method. 69. Simulatedmoving bed chromatography (SMBC) 79 Limitations of preparative elution chromatography: 1. Excessive consumption of solid and mobile phases 2. Discrete, interrupted process SMBC process has made liquid chromatography economically feasible on an industrial scale due to its high productivity relative to batch methods (up to 20-fold) 70. Simulatedmoving bed chromatography (SMBC) 80 71. Simulatedmoving bed chromatography (SMBC) 81 72. Simulatedmoving bed chromatography (SMBC) 82 73. Simulatedmoving bed chromatography (SMBC) 83 74. Simulatedmoving bed chromatography (SMBC) 84 75. Simulatedmoving bed chromatography (SMBC) 85 76. Simulatedmoving bed chromatography (SMBC) 86 Octave Chromatography System 77. Simulatedmoving bed chromatography (SMBC) 87 Benefits Drawbacks Continuous Higher investment cost compared to single column operations Higher recovery and purity Higher complexity Lower solvent consumption Higher maintenance costs Higher productivity up to 20-fold vs. batch systems Not ideal for the isolation of a single component or a fraction out of a multicomponent mixture Larger stationary phase particles, lower pressures Not readily suited for solvent gradients Scalable from milligrams to tons of purified product - 78. Simulatedmoving bed chromatography (SMBC) 88 Separation of a pharmaceutical intermediate racemate mixture on a chiral stationary phase (CSP) Miller et al. Journal of Chromatography A 1006.1 (2003): 267-280. 79. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 89 80. Twin-column CaptureSMB The fact that several mAbs are about to lose patent exclusivity over the next eight years, it is driving the biopharmaceutical industry toward more efficient and cost- effective downstream process. Protein A affinity chromatography is often employed for capture of mAbs and Fc fusion proteins from cell culture supernatants due to its capability of delivering product with high purity, yield and throughput. Although its price is almost one order of magnitude higher compared to non-affinity materials [12], no alternative processes that reach comparable purity levels have been established in industry. The CaptureSMB process has been developed to optimize the processing time and to reduce the consumption of expensive Protein A resin material in a production context. 90Angarita et al. Journal of Chromatography A 1389 (2015): 85-95. 81. Twin-column CaptureSMB The process uses twin columns, where one column is fully loaded and captures the breakthrough of the second column. Therefore it can be loaded faster than a single column process. The fully loaded column is then washed, the product eluted and the column is regenerated before being placed after the second column that is loaded until breakthrough, etc. This cyclic process allows the full use of the capture resin, whereas in a single column batch capture, 40-60% of resin material is never used due to the shape of the breakthrough curve. Automated process development and process control through a ChromIQ software module. CaptureSMB can save 2.5 mio USD p.a. COGs at production scale. 91Angarita et al. Journal of Chromatography A 1389 (2015): 85-95. 82. Twin-column CaptureSMB Adopted from: http://geproteinskills.blogspot.co.uk/2014/10/resins-protein-binding-capacities.html 83. Twin-column CaptureSMB 93Angarita et al. Journal of Chromatography A 1389 (2015): 85-95. 84. Twin-column CaptureSMB 94 85. Feed column length Internal concentration t1 t2 t3 Feed Wasted resin capacity in batch chromatography Twin-column CaptureSMB 95 86. Twin-column CaptureSMB 96 87. Feed Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 97 88. Feed Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 98 89. Wash Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 99 90. Elution Feed Product Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 100 91. Feed CIP & Re-equilibration Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 101 92. Feed Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 102 93. Wash Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 103 94. Elution Product Feed Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 104 95. CIP & Re-equil. Feed Step 1: Feed & wash Step 2: elute product feed column Step 3: CIP & re-equil. feed column Twin-column CaptureSMB 105 96. Twin-column CaptureSMB Thus CaptureSMB advantage: Increased resin utilization of 1st column. CaptureSMB process shows significant advantages in terms of loading (capacity utilization), productivity and buffer consumption in comparison to batch processes. 106Angarita et al. Journal of Chromatography A 1389 (2015): 85-95. 97. Twin-column CaptureSMB The product concentration is higher for CaptureSMB processes compared to batch processes while the product quality remains comparable. 107Angarita et al. Journal of Chromatography A 1389 (2015): 85-95. 98. Outline Introduction Novel materials 1. Mix&Go 2. Environment-responsive matrices 3. Novel fusion tags 4. Ionic liquid polymers 5. Monolithic cryogels 6. Other novel stationary phases Novel techniques/instruments 1. Single-Use Technology in Downstream Processing 2. Stirred-well filtration (SWF) 3. Simulated moving bed chromatography (SMBC) 4. Twin-column Capture SMB Summary 108 99. Lets sum it up! There are ongoing innovative inventions trying to be game-changing However, chromatography is still the workhorse of bioseparation. Streamlining and optimization of the current methods is vastly being studied. Here, the most recent ones were collected to give a view. Continuous chromatography is a great leap! 100. Thank you