1) The document describes a study to develop a continuous gradient assisted hydrophobic interaction chromatography process using Simulated Moving Bed (SMB-HIC) for the purification of recombinant streptokinase (rec-stk) from E. coli cell homogenate.
2) Key steps included producing and purifying rec-stk, estimating adsorption isotherms, designing a 3-zone open loop SMB system using a scanning program, and experimental realization of the SMB process.
3) Simulation studies were conducted to identify separation regions using equilibrium theory and stage models, and operating points were selected for experimental validation of the continuous purification of rec-stk via SMB-HIC.
The document describes tests using a Membrane Interface Probe with Heated Trunkline to measure the response of a Photoionization detector and Halogen specific detector to varying concentrations of trichloroethene. Graphs show the detector responses over time to TCE standards of 50-400 ppm, and the peak detector responses to concentrations from 150 ppb to 700,000 ug/L. The purpose is to demonstrate the instrument's ability to detect different TCE concentrations under controlled laboratory conditions.
The document describes tests using a Membrane Interface Probe with Heated Trunkline to measure the response of a Photoionization detector and Halogen specific detector to varying concentrations of trichloroethene. Graphs show the detector responses over time to TCE standards of 50-400 ppm, and the peak detector responses to concentrations from 150 ppb to 700,000 ug/L. The purpose is to demonstrate the instrument's ability to detect different TCE concentrations under controlled laboratory conditions.
This document discusses protein-protein interactions and how to distinguish specific from non-specific interfaces. It finds that:
1) Specific interfaces tend to be larger in size (>800 Å2) and more closely packed than non-specific interfaces like crystal packing.
2) Specific interfaces bury more atoms (>30% buried) and have a higher non-polar fraction than non-specific interfaces.
3) Parameters like fraction of buried atoms, non-polar area, and residue propensity score can identify homodimers and distinguish specific from non-specific interfaces with 95% accuracy. Combining structural features provides rules for determining interaction specificity.
This document summarizes experimental work validating numerical simulations of precise polymer melt processing using the Cambridge Multipass Rheometer (MPR). [1] The MPR was used to perform slit flow and cross-slot flow experiments on polymer melts like polystyrene. [2] Both the Pom-Pom and Rolie-Poly constitutive models were able to simulate the rheology and match experimental pressure drop and flow patterns when appropriate relaxation spectra and nonlinear parameters were used. [3] The validation demonstrates the ability of numerical simulation to accurately model complex viscoelastic flows.
This document summarizes a study on more environmentally friendly wood preservatives and coatings. Researchers tested boron silicates, copperazole, and CCA preservatives as well as varnish, stain, and dye coatings on radiata pine wood. Boron silicates and copperazole showed higher retention rates and less corrosion than CCA. Stain coatings performed best and had lower costs than varnish. Treatment costs were similar for boron silicate and copperazole wood coated with stain, providing environmentally-friendly and cost-effective wood protection alternatives.
The document discusses UltraPerformance Convergence Chromatography (UPC2) which utilizes liquid carbon dioxide as a mobile phase to provide increased selectivity compared to liquid and gas chromatography. UPC2 leverages the principles of normal phase chromatography with the ease of use of reversed-phase liquid chromatography. The ACQUITY UPC2 System delivers reliability, robustness, sensitivity and throughput for this technique. UPC2 offers unique benefits for applications in pharmaceuticals, food and beverage, chemicals and more.
3D Perfusion Bioreactor Technical Presentation3D Biotek
The 3D Perfusion Bioreactor™ is manufactured by 3D Biotek, LLC, an innovative technology company based in North Brunwick, New Jersey. 3D Biotek launched the 3D Perfusion Bioreactor™ in 2011, as a novel alternative to integrate the need for innovative cellular maintenance and increasing trends towards 3-dimensional cell culture. The 3D Perfusion Bioreactor is engineered to perfuse porous 3D polymer Inserts™ in 4 separate polycarbonate chambers able to fit up to 10-scaffolds per chamber. The 3D Insert has been validated by NIST (National Institute of Standards) for its open-pore, ample surface area, and 100% interconnected geometry, important features for direct application in scale-up processes, from cell expansion to recombinant protein production.
This document summarizes research on two proteins - Human Retinol Binding Protein-4 (RBP4) and Kvβ2, a subunit of the Kv1 potassium channel. For RBP4, the researcher optimized PCR conditions to amplify the gene and assessed RBP4's proposed role in insulin resistance and diabetes. For Kvβ2, the researcher overexpressed and purified the protein, then tested the inhibitory effects of rutin, quercetin, and resveratrol on Kvβ2 activity. The results provide new insights into physiological processes involving the Shaker potassium channel and identify resveratrol as a potential inhibitor of Kvβ2 activity. Overall, the research yielded beneficial PCR guidelines
The document describes tests using a Membrane Interface Probe with Heated Trunkline to measure the response of a Photoionization detector and Halogen specific detector to varying concentrations of trichloroethene. Graphs show the detector responses over time to TCE standards of 50-400 ppm, and the peak detector responses to concentrations from 150 ppb to 700,000 ug/L. The purpose is to demonstrate the instrument's ability to detect different TCE concentrations under controlled laboratory conditions.
The document describes tests using a Membrane Interface Probe with Heated Trunkline to measure the response of a Photoionization detector and Halogen specific detector to varying concentrations of trichloroethene. Graphs show the detector responses over time to TCE standards of 50-400 ppm, and the peak detector responses to concentrations from 150 ppb to 700,000 ug/L. The purpose is to demonstrate the instrument's ability to detect different TCE concentrations under controlled laboratory conditions.
This document discusses protein-protein interactions and how to distinguish specific from non-specific interfaces. It finds that:
1) Specific interfaces tend to be larger in size (>800 Å2) and more closely packed than non-specific interfaces like crystal packing.
2) Specific interfaces bury more atoms (>30% buried) and have a higher non-polar fraction than non-specific interfaces.
3) Parameters like fraction of buried atoms, non-polar area, and residue propensity score can identify homodimers and distinguish specific from non-specific interfaces with 95% accuracy. Combining structural features provides rules for determining interaction specificity.
This document summarizes experimental work validating numerical simulations of precise polymer melt processing using the Cambridge Multipass Rheometer (MPR). [1] The MPR was used to perform slit flow and cross-slot flow experiments on polymer melts like polystyrene. [2] Both the Pom-Pom and Rolie-Poly constitutive models were able to simulate the rheology and match experimental pressure drop and flow patterns when appropriate relaxation spectra and nonlinear parameters were used. [3] The validation demonstrates the ability of numerical simulation to accurately model complex viscoelastic flows.
This document summarizes a study on more environmentally friendly wood preservatives and coatings. Researchers tested boron silicates, copperazole, and CCA preservatives as well as varnish, stain, and dye coatings on radiata pine wood. Boron silicates and copperazole showed higher retention rates and less corrosion than CCA. Stain coatings performed best and had lower costs than varnish. Treatment costs were similar for boron silicate and copperazole wood coated with stain, providing environmentally-friendly and cost-effective wood protection alternatives.
The document discusses UltraPerformance Convergence Chromatography (UPC2) which utilizes liquid carbon dioxide as a mobile phase to provide increased selectivity compared to liquid and gas chromatography. UPC2 leverages the principles of normal phase chromatography with the ease of use of reversed-phase liquid chromatography. The ACQUITY UPC2 System delivers reliability, robustness, sensitivity and throughput for this technique. UPC2 offers unique benefits for applications in pharmaceuticals, food and beverage, chemicals and more.
3D Perfusion Bioreactor Technical Presentation3D Biotek
The 3D Perfusion Bioreactor™ is manufactured by 3D Biotek, LLC, an innovative technology company based in North Brunwick, New Jersey. 3D Biotek launched the 3D Perfusion Bioreactor™ in 2011, as a novel alternative to integrate the need for innovative cellular maintenance and increasing trends towards 3-dimensional cell culture. The 3D Perfusion Bioreactor is engineered to perfuse porous 3D polymer Inserts™ in 4 separate polycarbonate chambers able to fit up to 10-scaffolds per chamber. The 3D Insert has been validated by NIST (National Institute of Standards) for its open-pore, ample surface area, and 100% interconnected geometry, important features for direct application in scale-up processes, from cell expansion to recombinant protein production.
This document summarizes research on two proteins - Human Retinol Binding Protein-4 (RBP4) and Kvβ2, a subunit of the Kv1 potassium channel. For RBP4, the researcher optimized PCR conditions to amplify the gene and assessed RBP4's proposed role in insulin resistance and diabetes. For Kvβ2, the researcher overexpressed and purified the protein, then tested the inhibitory effects of rutin, quercetin, and resveratrol on Kvβ2 activity. The results provide new insights into physiological processes involving the Shaker potassium channel and identify resveratrol as a potential inhibitor of Kvβ2 activity. Overall, the research yielded beneficial PCR guidelines
Bioprocessing makes use of surface area such as packed beds and hollow fibers for scale up applications. In the same manner 3D porous scaffolds can serve the purpose to deliver both ample surface area and volumetric growth. Here, we present the 3D Perfusion Bioreactor.
Deformulating Complex Polymer Mixtures By GPC-IR Technologymzhou45
This document summarizes a presentation on using hyphenated GPC-IR technology to de-formulate complex polymer mixtures. The presentation discusses the DiscovIR-LC system and its features for GPC-IR and HPLC-IR analysis. It provides several case studies demonstrating how GPC-IR can be used to identify individual polymer components, characterize copolymer compositions, analyze polymer additives and degradation, and more. The speaker concludes that GPC-IR is a powerful tool for de-formulating complex polymer systems and problem solving in various industries.
The document describes tools and methods for screening compounds to find effective drugs for anxiety and depression. A typical screening process involves chemical synthesis of compounds, followed by screening assays to test binding and functional activity. Data is analyzed to select hit compounds, which then undergo further testing through ex vivo occupancy studies, in vitro ADME assays, and in vivo safety and efficacy models. Key methods discussed include radioligand binding assays to test if compounds compete for the same binding site as labeled ligands, and saturation binding assays to determine receptor affinity (Kd) and maximum binding (Bmax). Comparing results between species and receptor subtypes can provide insights into a compound's potential and selectivity.
ACS2010 GPC-IR To Characterize Macromolecular Excipients In Pharmaceutical ...mzhou45
The document summarizes a presentation given at the 2010 ACS National Meeting on using GPC-IR (gel permeation chromatography coupled with infrared spectroscopy) to characterize macromolecular excipients in pharmaceutical formulations. Specifically, it discusses using GPC-IR to analyze compositional drifts in copolymers like copovidone and degradation of excipients like HPMCAS and Eudragit L100-55 during hot melt extrusion processes. The technique allows examining changes in functional groups across the molecular weight distribution of excipients.
Cross-linked acrylic polymers are high molecular weight acrylic acid polymers cross-linked with allyl ethers to form a tightly coiled dry state. When dispersed in water, they uncoil slightly and thicken solutions minimally. Neutralization of carboxylic acid groups causes dramatic uncoiling and thickening at concentrations below 0.5%, making them highly efficient rheology modifiers. They are recommended for use in pharmaceutical, personal care, household, and industrial products and solutions, with different grades available depending on viscosity needs.
M A S S T R A N S F E R O P E R A T I O N S I J N T U M O D E L P A P E...guest3f9c6b
This document contains questions from a Mass Transfer Operations exam. It includes 8 questions related to various mass transfer topics like classification of mass transfer operations, diffusion, distillation design, and mass transfer correlations. The questions involve calculations related to diffusion rates, mass transfer coefficients, distillation column design parameters, and phase equilibrium data.
This document provides an overview of robots and automation equipment from a company. It includes:
- Short descriptions and key specifications of 10 different robot models ranging from 3kg to 60kg payloads and 0.58m to 2.55m reaches.
- A list of 10 reasons for manufacturers to invest in robots, including reducing costs, improving quality and safety, and increasing production outputs.
- An index listing other automation equipment like controllers, track motions, application equipment, painting robots, positioners, and software products.
EarthLed LumiSelect™ MR16 Pro Lamp - 7 wattEarthLED
The first dimmable MR-16 (GU5.3) that can replace 50 to 60 watt halogen lamps. LumiSelect™ MR-16 PRO offers a no compromise solution where a high output MLV dimmable light source is needed. Its compact dimensions mean it can be installed in any MR-16 socket, even units that still have UV filter retention clips. Best of all, the LumiSelect™ MR-16 PRO only consumes 7 watts of power.
This product can provide synergistic processing and cured resin performance advantages relative to simple mixtures of polyetheramines (PEAs) and cycloaliphatic amines.
The document outlines an experimental setup to develop a 3D ex-vivo model of chronic lymphocytic leukemia (CLL) using polyhydroxyalkanoate (PHA) scaffolds. The study aims to fabricate porous 3D scaffolds over 2mm thick using solvent casting and particulate leaching (SCPL) with PHB and PHBV. Initial results show that 4% polymer concentrations produced the thickest scaffolds of up to 10mm. Conductivity measurements supported the efficacy of the SCPL technique. Future work will optimize scaffold properties and evaluate their ability to culture leukaemic cells ex vivo.
17 leco's citius high res lcms-faster results-fewer runs david schlegel - lec...CPSA-2012_5-Minutes-Fame
The document discusses LECO's Citius High Res LC/MS instrument. It can (1) produce qualitative and quantitative results from a single run on the same instrument, (2) obtain excellent results faster with fewer runs through high resolution time-of-flight mass spectrometry, and (3) provide researchers a single instrument that can produce high quality results in a single analysis through post-acquisition data processing.
1. Researchers transformed green algae cells that grow through photosynthesis (autotrophically) into yellow cells that grow by consuming organic carbon (heterotrophically). This resulted in the altered cells accumulating much higher levels of lipids.
2. The lipid-rich heterotrophic cells were then used to produce biofuels through two methods - fast pyrolysis and biodiesel production. Both yielded higher quantities and quality of biofuels compared to the autotrophic cells.
3. The study demonstrates an effective process that combines biotechnology and engineering to optimize microalgae for high-yield biofuel production, which could have great commercial potential for liquid fuel production.
This document describes the TOYOPEARL AF-rProtein A HC-650F resin for purification of monoclonal antibodies. It has the highest dynamic binding capacity of commercial protein A resins, with 70 g/L capacity for IgG at 5 minutes residence time. The recombinant protein A ligand is alkali resistant, allowing over 200 cleaning-in-place cycles with 0.1M NaOH. Testing on a therapeutic monoclonal antibody showed binding capacities remained close to 50 mg/mL even at short residence times of 45 seconds.
HBOT may help reduce cognitive decline after CABG surgery and protect cells against stress. HBOT stimulates vascular tube formation, protects cells against heat shock, and regulates gene expression related to oxidative stress response. It increases expression of genes involved in cell proliferation, migration, and wound healing while decreasing expression of genes related to ER and Golgi pathways. HBOT promotes rapid recovery from injury through multiple protective and regenerative mechanisms.
Microbial synthesis of succinic acid from Typha grass hydrolysate and its application in biopolymer synthesis and as co-plasticizer. The document discusses using Actinobacillus succinogenes to ferment Typha grass hydrolysate to produce succinic acid. The succinic acid will then be used to synthesize polybutylene succinate-starch copolymer and glycerol-plasticized thermoplastic starch with succinic acid as a co-plasticizer through melt blending and extrusion. The polymers will be characterized and tested for physical, mechanical, and biodegradation properties.
Engineering Chemistry Jntu Model Paper{Www.Studentyogi.Com}guest3f9c6b
This document contains four sets of questions for an Engineering Chemistry exam. Each set contains 8 questions related to topics in engineering chemistry. The questions cover topics like water chemistry, corrosion, fuels, lubricants, polymers, coatings, and refractory materials. Students are instructed to answer any 5 of the 8 questions in each set, which vary in length from short answer to longer explanations and calculations.
Engineering Chemistry 1 Jntu Model Paper{Www.Studentyogi.Com}guest3f9c6b
This document appears to contain an exam for an Engineering Chemistry course, with 8 multi-part questions covering various topics in chemistry. It provides the exam code, date, course details, instructions to answer 5 of the 8 questions, and then lists the 8 questions which cover topics such as water analysis, corrosion, fuels, polymers, lubrication, and refractories. Each question has between 2-4 parts requiring explanations of concepts, processes, comparisons, or calculations.
The document summarizes steps taken to purify a fluorescent protein tag from a cyanobacteriochrome found in Thermosynechococcus elongatus, including:
1) Transforming E. coli with the gene for the cyanobacteriochrome tag and inducing protein expression.
2) Lysing the cells using a microfluidizer and centrifuging to remove cell debris.
3) Purifying the fluorescent tag protein using a chitin bead column.
4) Analyzing purity using SDS-PAGE gel electrophoresis and a spectrophotometer.
Expression Purification and Immunodetection of a fusion protein Glutathione S...iosrjce
Glutathione S Transferase(GST) is an enzyme of a multi gene family which is involved in reducing
oxidative damage to cells and detoxification of Xenobiotic compounds and plays critical role in life processes.
The entire work was completely qualitative and the objective of my work was to deal with the induction,
extraction and purification of the GST fusion protein from pGEX 3X vector.In order to achieve high degree of
transformed cells,the E.Coli BL21 host strain was made competent using 0.1M CaCl2 and adding of pGEX 3X
vector into host made it transformed.With the induction of GST protein by 0.1mM IPTG,the desired protein was
purified through glutathione Cl agarose column and was detected by immunoblotting method with the use of
anti GST HRP conjugate Ab which expressed the desired protein.
Bioprocessing makes use of surface area such as packed beds and hollow fibers for scale up applications. In the same manner 3D porous scaffolds can serve the purpose to deliver both ample surface area and volumetric growth. Here, we present the 3D Perfusion Bioreactor.
Deformulating Complex Polymer Mixtures By GPC-IR Technologymzhou45
This document summarizes a presentation on using hyphenated GPC-IR technology to de-formulate complex polymer mixtures. The presentation discusses the DiscovIR-LC system and its features for GPC-IR and HPLC-IR analysis. It provides several case studies demonstrating how GPC-IR can be used to identify individual polymer components, characterize copolymer compositions, analyze polymer additives and degradation, and more. The speaker concludes that GPC-IR is a powerful tool for de-formulating complex polymer systems and problem solving in various industries.
The document describes tools and methods for screening compounds to find effective drugs for anxiety and depression. A typical screening process involves chemical synthesis of compounds, followed by screening assays to test binding and functional activity. Data is analyzed to select hit compounds, which then undergo further testing through ex vivo occupancy studies, in vitro ADME assays, and in vivo safety and efficacy models. Key methods discussed include radioligand binding assays to test if compounds compete for the same binding site as labeled ligands, and saturation binding assays to determine receptor affinity (Kd) and maximum binding (Bmax). Comparing results between species and receptor subtypes can provide insights into a compound's potential and selectivity.
ACS2010 GPC-IR To Characterize Macromolecular Excipients In Pharmaceutical ...mzhou45
The document summarizes a presentation given at the 2010 ACS National Meeting on using GPC-IR (gel permeation chromatography coupled with infrared spectroscopy) to characterize macromolecular excipients in pharmaceutical formulations. Specifically, it discusses using GPC-IR to analyze compositional drifts in copolymers like copovidone and degradation of excipients like HPMCAS and Eudragit L100-55 during hot melt extrusion processes. The technique allows examining changes in functional groups across the molecular weight distribution of excipients.
Cross-linked acrylic polymers are high molecular weight acrylic acid polymers cross-linked with allyl ethers to form a tightly coiled dry state. When dispersed in water, they uncoil slightly and thicken solutions minimally. Neutralization of carboxylic acid groups causes dramatic uncoiling and thickening at concentrations below 0.5%, making them highly efficient rheology modifiers. They are recommended for use in pharmaceutical, personal care, household, and industrial products and solutions, with different grades available depending on viscosity needs.
M A S S T R A N S F E R O P E R A T I O N S I J N T U M O D E L P A P E...guest3f9c6b
This document contains questions from a Mass Transfer Operations exam. It includes 8 questions related to various mass transfer topics like classification of mass transfer operations, diffusion, distillation design, and mass transfer correlations. The questions involve calculations related to diffusion rates, mass transfer coefficients, distillation column design parameters, and phase equilibrium data.
This document provides an overview of robots and automation equipment from a company. It includes:
- Short descriptions and key specifications of 10 different robot models ranging from 3kg to 60kg payloads and 0.58m to 2.55m reaches.
- A list of 10 reasons for manufacturers to invest in robots, including reducing costs, improving quality and safety, and increasing production outputs.
- An index listing other automation equipment like controllers, track motions, application equipment, painting robots, positioners, and software products.
EarthLed LumiSelect™ MR16 Pro Lamp - 7 wattEarthLED
The first dimmable MR-16 (GU5.3) that can replace 50 to 60 watt halogen lamps. LumiSelect™ MR-16 PRO offers a no compromise solution where a high output MLV dimmable light source is needed. Its compact dimensions mean it can be installed in any MR-16 socket, even units that still have UV filter retention clips. Best of all, the LumiSelect™ MR-16 PRO only consumes 7 watts of power.
This product can provide synergistic processing and cured resin performance advantages relative to simple mixtures of polyetheramines (PEAs) and cycloaliphatic amines.
The document outlines an experimental setup to develop a 3D ex-vivo model of chronic lymphocytic leukemia (CLL) using polyhydroxyalkanoate (PHA) scaffolds. The study aims to fabricate porous 3D scaffolds over 2mm thick using solvent casting and particulate leaching (SCPL) with PHB and PHBV. Initial results show that 4% polymer concentrations produced the thickest scaffolds of up to 10mm. Conductivity measurements supported the efficacy of the SCPL technique. Future work will optimize scaffold properties and evaluate their ability to culture leukaemic cells ex vivo.
17 leco's citius high res lcms-faster results-fewer runs david schlegel - lec...CPSA-2012_5-Minutes-Fame
The document discusses LECO's Citius High Res LC/MS instrument. It can (1) produce qualitative and quantitative results from a single run on the same instrument, (2) obtain excellent results faster with fewer runs through high resolution time-of-flight mass spectrometry, and (3) provide researchers a single instrument that can produce high quality results in a single analysis through post-acquisition data processing.
1. Researchers transformed green algae cells that grow through photosynthesis (autotrophically) into yellow cells that grow by consuming organic carbon (heterotrophically). This resulted in the altered cells accumulating much higher levels of lipids.
2. The lipid-rich heterotrophic cells were then used to produce biofuels through two methods - fast pyrolysis and biodiesel production. Both yielded higher quantities and quality of biofuels compared to the autotrophic cells.
3. The study demonstrates an effective process that combines biotechnology and engineering to optimize microalgae for high-yield biofuel production, which could have great commercial potential for liquid fuel production.
This document describes the TOYOPEARL AF-rProtein A HC-650F resin for purification of monoclonal antibodies. It has the highest dynamic binding capacity of commercial protein A resins, with 70 g/L capacity for IgG at 5 minutes residence time. The recombinant protein A ligand is alkali resistant, allowing over 200 cleaning-in-place cycles with 0.1M NaOH. Testing on a therapeutic monoclonal antibody showed binding capacities remained close to 50 mg/mL even at short residence times of 45 seconds.
HBOT may help reduce cognitive decline after CABG surgery and protect cells against stress. HBOT stimulates vascular tube formation, protects cells against heat shock, and regulates gene expression related to oxidative stress response. It increases expression of genes involved in cell proliferation, migration, and wound healing while decreasing expression of genes related to ER and Golgi pathways. HBOT promotes rapid recovery from injury through multiple protective and regenerative mechanisms.
Microbial synthesis of succinic acid from Typha grass hydrolysate and its application in biopolymer synthesis and as co-plasticizer. The document discusses using Actinobacillus succinogenes to ferment Typha grass hydrolysate to produce succinic acid. The succinic acid will then be used to synthesize polybutylene succinate-starch copolymer and glycerol-plasticized thermoplastic starch with succinic acid as a co-plasticizer through melt blending and extrusion. The polymers will be characterized and tested for physical, mechanical, and biodegradation properties.
Engineering Chemistry Jntu Model Paper{Www.Studentyogi.Com}guest3f9c6b
This document contains four sets of questions for an Engineering Chemistry exam. Each set contains 8 questions related to topics in engineering chemistry. The questions cover topics like water chemistry, corrosion, fuels, lubricants, polymers, coatings, and refractory materials. Students are instructed to answer any 5 of the 8 questions in each set, which vary in length from short answer to longer explanations and calculations.
Engineering Chemistry 1 Jntu Model Paper{Www.Studentyogi.Com}guest3f9c6b
This document appears to contain an exam for an Engineering Chemistry course, with 8 multi-part questions covering various topics in chemistry. It provides the exam code, date, course details, instructions to answer 5 of the 8 questions, and then lists the 8 questions which cover topics such as water analysis, corrosion, fuels, polymers, lubrication, and refractories. Each question has between 2-4 parts requiring explanations of concepts, processes, comparisons, or calculations.
The document summarizes steps taken to purify a fluorescent protein tag from a cyanobacteriochrome found in Thermosynechococcus elongatus, including:
1) Transforming E. coli with the gene for the cyanobacteriochrome tag and inducing protein expression.
2) Lysing the cells using a microfluidizer and centrifuging to remove cell debris.
3) Purifying the fluorescent tag protein using a chitin bead column.
4) Analyzing purity using SDS-PAGE gel electrophoresis and a spectrophotometer.
Expression Purification and Immunodetection of a fusion protein Glutathione S...iosrjce
Glutathione S Transferase(GST) is an enzyme of a multi gene family which is involved in reducing
oxidative damage to cells and detoxification of Xenobiotic compounds and plays critical role in life processes.
The entire work was completely qualitative and the objective of my work was to deal with the induction,
extraction and purification of the GST fusion protein from pGEX 3X vector.In order to achieve high degree of
transformed cells,the E.Coli BL21 host strain was made competent using 0.1M CaCl2 and adding of pGEX 3X
vector into host made it transformed.With the induction of GST protein by 0.1mM IPTG,the desired protein was
purified through glutathione Cl agarose column and was detected by immunoblotting method with the use of
anti GST HRP conjugate Ab which expressed the desired protein.
Examples of Qualifying Techniques- Affinity Chromatography, SDS-Page, Gel Ele...Jacob Feste
This document summarizes a laboratory experiment to purify a protein using affinity chromatography and identify it using SDS-PAGE. Bacterial cells containing a plasmid for overexpressing the protein Riboflavin Kinase (NcRFK) were lysed and the lysate was purified using GSH-Agarose columns. Samples of lysate, flow-through, and purified protein were run on an SDS-PAGE gel. The results showed bands around 5-10 kDa for each sample, suggesting partial but not complete purification of NcRFK had occurred. Further mass spectrometry would be needed to fully determine purity. The objective of isolating NcRFK was thus partially but not fully achieved.
This document summarizes several common techniques used to purify and characterize proteins:
1. Proteins are first isolated from cells through homogenization and then separated from contaminants using techniques like salting out with ammonium sulfate or differential centrifugation.
2. Column chromatography techniques like size-exclusion chromatography, affinity chromatography, and ion exchange chromatography are used to further purify proteins based on properties like size, specific binding interactions, or charge.
3. Electrophoresis, isoelectric focusing, and techniques like amino acid analysis, Edman degradation, and enzymatic or chemical cleavage are then used to determine a protein's primary structure by separating peptide fragments and determining their sequence.
This document discusses various methods for purifying proteins, including selecting an appropriate source, lysing cells, centrifugation, chromatography, electrophoresis, and measuring protein activity. Key steps involve disrupting cells to release proteins, separating components by techniques that exploit differences in properties like size, charge, and affinity for ligands, and ensuring the purified protein retains its native structure and activity. The goal is to enrich the target protein away from other biomolecules to obtain a pure sample for downstream applications.
This document discusses various protein purification strategies and techniques. It outlines steps for protein isolation, extraction, concentration, and precipitation. It also describes different chromatography methods for purification, including gel filtration, ion exchange, hydrophobic interaction, and affinity chromatography. It provides details on analytical tools like SDS-PAGE for assessing purity and discusses considerations for combining purification techniques.
1. Recombinant protein purification using
gradient assisted Simulated Moving Bed-
Hydrophobic Interaction Chromatography
(SMB-HIC)
Sivakumar P.
BT04D006
PhD viva-voce
MAX-PLANCK-INSTITUT
Research Advisors: DYNAMIK KOMPLEXER
TECHNISCHER
Prof. Guhan Jayaraman
SYSTEME
MAGDEBURG
Prof.Andreas Seidel-Morgenstern
2. Overview
Background and objectives
Scheme for the work
Preparative work
Production and purification of recombinant streptokinase (rec-stk)
Adsorption isotherm estimation
SMB theory
Design of SMB
Scanning the separation zone
Simulation profiles
Experimental realization
Conclusion and Discussion
2
3. Back ground and Objective
Background
work at IIT-Madras
purification of rec-stk (batch chromatography*)
Objectives
Developing a continuous gradient assisted
HIC-SMB process for the purification of rec- stk
3
*B. Balagurunathan et al. / Biochemical Engineering Journal 39 (2008) 84–90
4. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Scheme for the work
Preparative work for SMB (batch)
Adsorption isotherm estimation
SMB Design
SMB Experiments
Data analysis
100L reactor SMB
HZI MPI
4
5. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Preparative work for SMB
rec-stk was produced in 100 L reactor and used as a feed for
HIC Matrices screening
Pure rec-stk production for adsorption isotherm estimation
SMB experiments
HIC matrices screening- Butyl sepharose
Selectivity
Binding capacity
Ease of regeneration
Preparative batch purification of rec-stk
5
6. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Adsorption equilibrium constants
(PULSE experiments)
14
STK
(NH4)2SO4 Contaminants STK STK+Degraded product
12
[mM] (Target)
KH,con1 KH,con2 KH,STK 10
50 2.23 2.23 8
H,i
100 1.59 3.69
˜ 0 6
K
150 1.52 7.76
4
200 1.57 11.74
2
qi K H ,i (Csalt )Ci i= degraded STK+STK,STK 0
0 50 100 150 200 250
C (NH [M]
4)2SO4
KH,i depend on Csalt approximately linearly between 100 and 200mM
6
Hi (Csalt ) a1,i a2,i (Csalt ) a1,im 1.59; a2,im 0.0002 a1, sk 4.34; a2, sk 0.081
7. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Introduction to continuous chromatography
Batch Chromatography
Species have different affinity towards adsorbent:
migrate with different velocities
leave column at different time points
S A A, B B True Moving Bed Chromatography
Analogy
Liquid and solid phase move in countercurrent:
Continuous operation.
Solid Phase Two outlets. Good for binary separations.
Difficult to realise due to the movement of the solid
phase.
Simulated Moving Bed
S
Raffinate Feed Extract
Practical realisation of the TMB concept: A A, B B
periodic switching of the columns
1 2 3 4 5 6
(or ports) 7
Periodic behaviour (cyclic steady state). 7
8. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
SMB theory
8
9. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Isocratic separation of binary mixture
(four zone SMB)
Lets consider
A+B mixture
Zone IV
A is weakly retained, B is strongly retained
&
mIV VIV Raffinate
A & follows linear Henrys isotherm model
, VR
Zone III Keys for separation
&
mIII , VIII
Feed &
&
Vi
&
VF mi
Zone II &
VS
A+B
&
mII , VII Extract i 1, 2, 3, 4
B
&
VE
Zone I
&
mI , VI Flow rate ratios mi
&
Vsolid m4 HA < H B m1
Desorbent
H A m2 H 9
B Triangle
H A m3 HB
Morbidelli et al., 1997
10. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Four zones closed loop SMB
Illustration of Triangle theory
HA Flow rate ratios mi
QI Safety value
mI HB
QS
4 2 6 HB QII
mII HA
QS
1 HB / H A 1
3 QIII
mIII HB /
QS
QIV
mIV HA /
QS
5 H A m2 H
B Triangle
H A m3 HB
HA After considering dead volume
10
Morbidelli et al., 1997
11. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Separation region (gradient SMB)
Linear isotherm
HA 1. Complete separation
2. Pure extract and raffinate polluted with species B
3. Pure raffinate and extract polluted with species A
2 HB 4. Both raffinate extract containing A and B
4 6
5. Extract flooded with desorbent (A&B at raffinate)
1 6. Raffinate flooded with desorbent (A&B at extract)
3
7. Extract flooded with desorbent (A accumulates)
8. Raffinate flooded with desorbent (B accumulates)
9. Raffinate and extract flooded and (A& B accumulates)
5
HA
After considering dead volume
11
Mazzotti et al., 1999, Abel et al., 2002
12. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of continuous separation process
3-zone open-loop two step-gradient SMB
12
13. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of 3-zone open-loop
two-step gradient process (TMB Model)
Raffinate: Two-step salt
contaminants, gradient
degraded &
VZ
streptokinase mz Z I , II , III
&
VS
Feed: E.coli cell
salt concentration
homogenate
containing
streptokinase
Extract:
streptokinase
Vcol (1 )
t* D R F
C salt Csalt C salt
&
VS 13
Gueorguieva L et al. J. Chromatogr. A, 1176,(2007) 69.
14. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of 3-zone open-loop gradient process
Parameters set: F D &
Csalt , Csalt , VF
&
VZ
Design parameters: mz Z I , II , III
&
VS
Inequalities for complete separation under gradient conditions
Henrys constants
D D
mI K H , STK
(C salt
) K H ,i K H ,i (Csalt ) i con, STK
D D D D D D
K H ,con (C salt ) 1.55
K H , con ( C salt ) m II K H , STK ( C salt )
D D
R R R R K H , STK (C salt ) 3.69
K H , con ( C salt ) m III K H , STK ( C salt ) R R
K H ,con (C salt ) 1.55
Feed D R R
R ( mIII mII )C salt mII C salt K H , STK (C salt ) 11.74
C salt
mIII 14
Rhee et al. (1970), Mazzotti et al. (1997)
15. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of 3-zone open-loop gradient TMB
Calculation of separation regions
(scanning program MPI)
1) Equilibrium theory Nz→ , Purity=99.99%
2) Equilibrium stage model NI= NII=NIII=20 , Purity=99%
16
F
C
1 . salt
Csalt
200mM , F
III
Csalt , D
D
m m II 14
C salt C
100mMsalt , F Csalt , R
12
& P2
VF 1mL/ min
2. H sk2R (Csalt , R )
L
,
*
10 L2
Value in zone 1 *
Csalt , R Csalt , D
PurityDconstraint H sk , D )
H sk , ( H sk , F
III
8
m
Csalt , F Csalt , D L1
6 lower
D D bound
K H ,con (C ) 1.55
salt C salt ,F C salt , R
*
II , L 2 *
3. m
D (C ) H sk2R
D
L 4
K H , STK (C ) 3.69 , C salt ,F C salt ,D
salt , R
salt P3
2
R R
K H ,con (C salt ) 1.55 Csalt ,F Csalt ,D P1
III , L 2 *
4. mR (CR ,R
salt ) m II ,L 2 * 0 15
K H , STK (C salt ) 11.74 Csalt ,F Csalt ,R 0 1 Him,D 2 3 Hsk,D4 5
II
m
16. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Separation region predictions from
scanning program
16
Separation regions
14
Thick lines: equilibrium theory
12 Symbols: predictions of
R2
equilibrium stage model
10
8 NI N II N III 50 I
2
III
m
R3 R1
6
R1: (○), Pusk ,E Puim,R 99%
4
R2: (∆), Pusk ,E 99%, Puim,R 70%
2
R3: (x), Pusk ,E 70%, Puim,R 99%
0
0 1 2 3 4 5
II
m 16
17. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experimental operating points
16
14
14
Operation line 175mM
b
14
b 1
12
12
12
P2
10
10
b
2 140mM
10 L2
88
Separation zone a
Operation
m III
8 b
3
III
m
L1
III
points lower
m
a
66
6 1
bound a
b
4 2
4 a
44 3 P3
a
2 4 Diagonal line
22 P1
0
00
0 1 Him,D 2 3 Hsk,D4 5
II
0,0
0,0 0,5
0,5 1,0
1,0 1,5
1,5 2,0
2,0 m2,5
2,5 3,0
3,0 3,5
3,5 4,0
4,0 4,5
4,5 5,0
5,0
II
II
m
m
17
18. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Parameters used for
simulation studies
Point I & & & &
Csalt , R m II m III t* VD VE VR VF
Nr. [mM] [-] [-] [-] [s] [mL/min] [mL/min] [mL/min] [mL/min]
1b 175 3.30 13.2 2 84 0.75 0.41 1.33 1
2b 175 2.64 10.6 2 125 0.93 0.60 1.33 1
3b 175 1.98 7.95 2 167 1.24 0.91 1.33 1
4b 175 1.32 5.3 2 208 1.86 1.53 1.33 1
1a 140 3.30 5.5 1.1 37 1.85 0.35 2.50 1
2a 140 2.64 4.4 1.1 28 2.32 0.82 2.50 1
3a 140 1.98 3.3 1.1 37 3.09 1.59 2.50 1
18
4a 140 1.32 2.2 1.1 46 2.32 1.57 1.25 0.5
19. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of internal profiles
(rec stk enriched) at 175mM raffinate salt concentration
R
C salt
= 175mM STK
30 b
CSTK= CIM = 5mg/mL 1
b
C
F
= 200mM 2
25 salt
b
C
D
salt
= 100mM 3
C STK, C IM [mg/ml]
VF = 1mL/Min
20
15
10
IM
Feed
concentration5
0
0.00 0.33 0.66 0.99
Desorbent Extract Feed Raffinate 19
Normalised Length
20. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experimental Validation
3-zone open-loop two-step gradient SMB
20
21. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
KNAUER (moving columns) precission valve from smb.mp4
CSEP C916 Multi-function valve Configuration
stator
rotor
Stationary phase Butyl HP
Mobile phase
Configuration
Desorbent
Three zones 1-1-1-(1)
phosphate buffer +100 mM (NH4)2 SO4
Each zone one 2 x1mL column
Feed
20 mM phosphate buffer+200 mM (NH4)2 SO4 UV product profile
Regeneration Internal UV profile and conductivity profile
Milli Q at flow rate of 3 mL/min. Cleaning zone conductivity and UV profile
21
SEC, SDS(PAGE) analysis
22. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experimental operating points
14
b
Operation line
b
1
12 175mM
b
10 2
8
140mM a
Separation zone
3
b
III
m
Operation a
6
points 1
b a
4 2
a
4 3
a
4
2 Diagonal line
0
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
II
m 22
23. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Parameters used for experiments
Point I & & & &
Csalt , R m II m III t* VD VE VR VF
Nr. [mM] [-] [-] [-] [s] [mL/min]
1b 175 3.30 13.2 2 84 0.83 0.39 1.42 1
2b 175 2.64 10.6 2 125 1.03 0.57 1.44 1
3b 175 1.98 7.95 2 167 1.37 0.87 1.48 1
4b 175 1.32 5.3 2 208 - - - 1
1a 140 3.30 5.5 1.1 37 2.21 0.24 2.89 1
2a 140 2.64 4.4 1.1 28 2.76 0.67 2.99 1
3a 140 1.98 3.3 1.1 37 3.68 1.40 3.15 1
23
4a 140 1.32 2.2 1.1 46 2.76 1.42 1.74 0.5
24. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
SDS-PAGE analysis of the
extract and raffinate outlet..
14
b
b 1
175mM
12
b
10 2
8
a 140mM
b
3
III
m
a
6 1
b a
4 2
a
4 3
a
4
Raffinate 2 Raffinate
salt concentration 140mM 0 salt concentration 175mM
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
II
4a 3a 2a 1a m
L 3b 2b 1b R-Raffinate
R E R E R E R E R E R E R E
E-Extract
L-Load
rec-stk
24
purity 73% 84% 78% 70% 77% 57% 56%
26. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experiments Vs simulation prediction
Point exp th exp th Yield
Csalt , R Csk ,E Csk ,E Pusk ,E Pusk ,E
Nr. [mM] [mg/mL] [mg/mL] [%] [%] [%]
1a 140 0.34 13.6 70 100 2
2a 140 0.50 6.07 78 100 8
3a 140 0.98 3.14 84 99.88 31
4a 140 1.42 1.60 73 78.83 93
1b 175 0.33 2.97 56.0 100 4
2b 175 0.31 6.15 57.0 100 6
3b 175 0.44 5.51 77 99.96 12
4b* 175 - 3.29 - 94.38 -
26
* Operation point 4b is not feasible because of the flow rate constraints
27. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Extract purity
Experimental Theoretical
100 100
Extract purity [%]
Extract purity [%]
80 80
60 60
40 40
20 20
0
0
1.0 1.5 2.0 2.5 3.0 3.5 4.0
1.0 1.5 2.0 2.5 3.0 3.5 4.0
II
m
II m
Closed squares for raffinate salt concentration 140mM
a) b) 27
Closed triangles for raffinate salt concentration 175mM
28. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Extract concentrations
Experimental Theoretical
2.0 14
Extract concentraiton [mg/mL]
Extract concentraiton [mg/mL]
12
1.5
10
8
1.0
6
4
0.5
2
0.0 0
1.0 1.5 2.0 2.5 3.0 3.5 4.0 1.0 1.5 2.0 2.5 3.0 3.5 4.0
II II
m m
Closed squares for raffinate salt concentration 140mM
Closed triangles for raffinate salt concentration 175mMb)
a) 28
29. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Enhancing the yield of the process
Effect of safety values (β factor)
14 SDA-PAGE gel analysis
12
b 175mM
1
b
b
10 2
8
a 140mM
b
III
m 3
a
6 1
b a
4 2
a
4 3
a
4
2
0
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 R E
β =1.1
II
m
S. Operational β =1.1 β =2 R- Raffinate
NO. point 2a E- Extract
1 Load (mg/mL) 17.2 17.2
2 Raffinate (mg/mL) 5.94 6.65
3 Extract (mg/mL) 0.34 0.20
4 Purity of stk (%) 69.8 69.4 29
R E
5 Recovery (%) 1.5% 6.7% β =2
30. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Reprocessing the Raffinate
(isocratic run at 140mM raffinate salt concentration )
14 14
13 13
12 12
11 11
10 10
9 9
8 8
mIII 7 7
6 6
5 5
4 4
3 3 β =1.1 β =2
2 2
1 1 R E R E L
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12
mII
S.NO β =1.1 β =2
1 Load (mg/mL) 6.76 6.76
2 Raffinate (mg/mL) 2.69 3.12 R- Raffinate
3 Extract (mg/mL) 0.38 0.20 E- Extract
L- Load (raffinate from SMB)
4 Purity of stk (%) 79 84 30
5 Recovery (%) 38 42
31. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Conclusions..
Butyl sepharose matrice found to be suitable for the continuous HIC-SMB
purification of rec-stk.
Adsorption isotherms for rec-stk with butyl sepharose matrice found to be:
linear adsorption isotherm, parameters depend on the salt
concentration.
perturbation method applied to evaluate isotherm linearity.
3 zone open- loop two- step gradient SMB design was proposed and
designed.
31
32. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Conclusions
Scanning program based on the equilibrium model and the equilibrium stage
model was employed to predict the separation zone.
Experimental realisation of the continuous purification of rec-stk.
Simulation predicts for more idealistic conditions and correlate with product
purity trends.
Highest product purity=84%(3a), and concentration of =1.4mg/mL with a mass
&
m flux of =2mg/min for operation point 4a
Recycling the raffinate will increase the yield of the process
32
33. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Discussion
Demonstrate the potential of SMB for the continuous recombinant protein
purification.
SMB could be placed for the high throughput capture and intermediate
purification of the recombinant protein from complex mixtures.
Fractionation and Feed back SMB or Tandem chromatography could be
proposed for increasing the yield of the purification process
33
34. presentations
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Determination of adsorption isotherm
parameters from total protein mixtures” PREP 2009 meeting, July 19-22, 2009, Loews philadelphia, USA(oral).
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Determination of adsorption isotherm
parameters for recombinant streptokinase using perturbation method” 5th Doktorandenseminar Präparative
chromatographie, March 1-3,2009, Wetter Ruhr, Germany(oral).
Palani S., Gueorguieva L., Rinas U., Jayaraman G., Seidel-Morgenstern A., “Simulated Moving Bed Chromatography
(SMBC) current status and applications for protein purification”(oral presentation), International symposium Gene to vial
concept for the biotechnology based health care molecules, Feb 7-10, 2010, VIT, Vellore, T.N, India.(oral).
Palani S., Jayaraman G., Gueorguieva L., Kessler L C., Rinas U., Seidel-Morgenstern A., “Continuous separation of
Recombinant streptokinase using hic gradient simulated moving bed chromatography”, 28th International Symposium on the
separation of Proteins, Peptides and Polynucleotide’s (ISPPP 2008), September 21-24, 2008, Baden-Baden,
Germany(poster).
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Continuous simulated moving bed (SMB)
purification of recombinant streptokinase”, 34th International Symposium on High Performance Liquid Phase Separations
and Related techniques, HPLC 2009,June 28-July 2, 2008, Dresden, Germany(poster).
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Kontinuierliche aufreinigung der
rekombinanten streptokinase mittels simulated moving bed (SMB) chromatographie”, 27th DECHEMA Jahrestagung der
34
Biotechnologen, gemeinsam mit International workshop on downstream processing. September 8-10, 2009, Mannheim,
Germany(poster).
35. Publications
Palani S., Gueorguieva L, Rinas U., Seidel-Morgenstern A., Jayaraman G., “ Continuous
purification of recombinant streptokinase using Hydrophobic Interaction- Gradient assisted
Simulated moving Bed Chromatography. part I. Determination of adsorption isotherms
applying perturbation method ”(Journal of chromatography A., manuscript submitted).
Gueorguieva L., Palani S., Rinas U., Jayaraman G.,Seidel-Morgenstern A.,“ Continuous
purification of recombinant streptokinase using Hydrophobic Interaction- Gradient assisted
Simulated moving Bed Chromatography. Part II. SMB experimental design analysis for the
operating conditions”(Journal of chromatography A., manuscript submitted).
35
36. Acknowledgement
Prof Guhan Jayaraman
Herr.Prof Andreas Seidel-Morgenstern, MPI,Magdeburg
Frau Ludmila Guorguieva, Dr.Christian Kessler,MPI
Frau Dr. Rinas, HZI, Braunschweig
Herr Dr. Wilko, IFN, Magdeburg
Collegues at IITM,MPI,IFN,HZI
IITM,Deutscher Academic Austausch Dienst (DAAD),
GDCh, Deutsche Forschungemeinschaft (SFG-578)
still lots needs to be explored with SMB!!!
36
40. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Classical True Moving Bed (TMB) chromatography
B
C Zone IV
S Zone IV
M
mIV V&IV
&
mIV VIV Raffinate
Raffinate
A &
A &
,, VR
VR
C
S
M
B
Zone III
Zone III
mIII ,,V&III
&
mIII VIII&&
Feed
Feed
&
&
VF
VF S
M
B
C
A+B
A+B Zone II
Zone II
mII ,,V&II
mII VII&
Extract
Extract
B
B
M
B
C
S
Zone II
Zone
V&E
&
VE
mII,,V&I
m VI &
&
&
Vsolid
Vsolid
Desorbent
Desorbent
40
41. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Adsorption isotherm estimation
Dynamic methods
Method and Special favorable feature Special Applicable for
characterization unfavorable more than one
feature solute
Perturbation No detector calibration Isotherm Difficult
(Dynamic, small samples) required model required
Dispersed front analysis Low sample amount, High column No
(ECP) small number of efficiency
(dynamic, intermediate experiments required
samples)
Chromatogram fitting Low sample amount, Models for the Difficult
(dynamic) small number of isotherms and
experiments to simulate the
chromatogram
41
required
Seidel-Morgenstern A, J. Chromatogr. A 1037,(2004) 255
42. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Adsorption isotherms
• Relationship between the
equilibrium protein
concentration in the
stationary phase and the
protein concentration in the
mobile phase
• Slope gives the information
about the affinity
• Plateau gives the information
about the capacity
42
43. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
SMB under linear and non-linear conditions
Regions 1 to 4 correspond to higher and higher feed concentrations
43
44. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Effect of feed concentration over flow rate ratios
44
49. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Motivation for perturbation method
• Commercial preparations
• Presence of Bovine Serum Albumin (BSA) as stabiliser
• Standards from NIBSC
• Smaller in quantity
• Over expression and purification
•Storage and degradation problem
• Perturbation method
• Crude homogenate as the feed material
49
Blumel C, Hugo p, Seidel Morgenstern P, (1999) J. Chromatogr. A 865 (1999)51
Heuer C, Kusers E, Plattner T, Seidel-Morgenstern A, J. Chromatogr. A 827 (1998)175.
50. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Retention time and adsorption isotherm
parameters
6
8
STK+degraded product
5
STK
Streptokinase
4 6
100 mM
tR, i [min]
150 mM
3 200 mM
Contaminants 4
KH,i
2 100 mM
150 mM
200 mM
1 2
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
Ci [mg/ml] 0 50 100 150 200 250
C(NH )2SO4
[M]
4
linear isotherms; KHi depend on Csalt
qi K H ,i (Csalt )Ci
50
i= degraded STK+STK,STK
51. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
True Moving bed chromatography-Analogy
Stationary phase
Mobile Phase
51
52. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of binary separation
52
53. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of binary separation
(Fast solid flow)
53
54. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of binary separation
Slow solid flow
54
55. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Bioreactor production of rec-stk
(HZI, Braunschweig)
Preparation of the 10L Inoculums preparation Preparation for the Reactor
(100L) reactor
Inoculation
Optical Density(OD) 4
Product analysis
Induction Fermentation Harvesting and Storage of cell pellet
55
with IPTG (4 hours) centrifugation in -80 ˚C
56. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Bioreactor Production of rec-stk
10 L Bioreactor
S.No Parameters Fermentor SDS PAGE analysis
1 Agitator speed 400-1000 in cascade rec-stk
mode 1 2 3 4 5
2 Temperature 37 Degree Celsius
3 Aeration 5 LPM 1 un-induced sample
4 Inoculum volume 2%
to fermentation 2 after induction (1.20 hours)
volume
3 after induction (2.20 hours)
5 IPTG 0.1mM
concentration
4 after induction (3.30 hours)
6 Induction OD 4 5 after induction (5 hours)
7 Fermentation 11 hours
duration (total)
8 Wet biomass 260 grams (10L)
produced 2.8kG (100L)
56
57. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
HIC matrices screening
Screening criteria
• binding conditions at 250,500,750mM
•Selective binding for the target protein
•Step (or) linear elution
Positive candidates
•Ease of regeneration
Phenyl sepharose 57
Butyl sepharose
58. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
HIC matrices screening
Phenyl sepharose – more hydrophobic streptokinase tails all over the gradients
Butyl sepharose - weakly hydrophobic
Binding capacity is higher for stk at low salt concentration
More selective binding of stk and easy regeneration
Manufacturers
recommendations
1 Recommended flow rate 1mL/Min
2 Maximum flow rate 4mL/Min
3 Column dimensions 0.7X2.5 cm
4 Column volume 1mL
5 Maximum backpressure 3 bar, 42psi, 0.3Mpa 58
59. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Preparative purification of recombinant
streptokinase
2 46 9 1115 21 23 35
3500
100
Absorbance at 280 nm (mAu)
Modifier Concentration (%B)
3000
80
2500
2000 60
1500
40
1000
20
500
0 0
0 20 40 60 80 100 120 140 160 180
Volume (mL) Absorbance at 280 nm (mAu)
Conductivity (mS/cm)
Modifier concentration (%B)
Feed : Total protein mixture from E.coli homogenate
Solid Phase : Butyl sepharose HP, GE Biosciences
L= 12cm; D= 0.5 cm,
Vcol= 9.5mL; dpart= 34 µm
Ligand : Butyl, 10 μmol/mL,
Mobile Phase : Buffer A: 20 mM sodium phosphate buffer + 0.2M (NH4)2S04 (pH 7.2)
59
Elution buffer : Buffer B: 20 mM sodium phosphate buffer (pH 7.2)
Continuation of the work IITM expertiseMpi expertise
Y=mx +c and the values given hereWithin this 100 -200 mm Separation is assumed to be pseudo binary separation
Mark the areas with number so that it is easy to explainExpalin why it is called triangleAnd mark the triangle hereWhat is the case with non linnear chromatographyHigher feed concentration
T star is the switch time in the smb unitQj is the fluid flow rate in the smb unitεb and εp are the bed void fraction and particle porosityε* is the overall bed void fraction ε* =εb +(1-εb)εpV is the column volumej-=1,2,3,4
Show the scanning prog here
Explain that same points were chosen for the experiments also
What is NIBSC
Easy to connect two columns togatherAdvantagesGood at higher flowrate maximum of 4ml per minute
What is NIBSC
So that one need not adjust the salt concentration
Talk about the assumptions Talk about the other namesTalk about the disadvantagesTalk about the way it is done