Sắc ký sinh học tinh sạch protein

ới Histidine. „ Recombinant Tags có thể được sử dụng để lợi dụng điều này (6 His tags) his his his his hishis Ni+ Tóm tắt về sắc ký ái lực Kỹ thuật hấp thụ sử dụng ái lực sinh hay hóa học mà protein có để cho một phân tử khác được cố định lên một phase ổn định. Ưu điểm ‟ Chuyên biệt ‟ độ tinh khiết cao trong một bước thực hiện ‟ Dễ dàng phát triển các phương pháp tinh sạch MAb phát triển trên Protein A ‟ Loại đựơc protein biến tính và chưa biến tính trong một số trường hợp. Khuyết điểm ‟ Đắt tiền ‟ Khó rửa sạch ‟ Lọc ligand ‟ Gắn ligand vào các môi trường hoạt tính không dễ ‟ Tạo ligand thường khó khăn. Strategy of Purification Fractionation procedures or steps to isolate protein based on physical characteristics. Characteristic Procedure •Charge 1. Ion exchange 2. Electrophoresis 3. Isoelectric focusing •Polarity 1. Adsorption chromatography 2. Paper chromatography 3. Reverse phase chromatography 4. Hydrophobic interaction Characteristic Procedure •Size 1. Dialysis and ultrafiltration 2. Gel electrophoresis 3. Gel filtration 4. Ultracentrifugation •Specificity 1. Affinity chromatography 2. Immunopurification •Solubility 1. Salt precipitation 2. Detergent solubilization What Chromatography Systems Bio-Rad can provide you for your application? • Econo Gradient Pump • Biologic LP System • The Biologic Duo-Flow System The Econo Gradient Pump (EGP) The Econo Gradient Pump • Ideal system for labs new to protein purification. • Ideal system for affinity chromatography of recombinant proteins. • Ideal for budget-minded customers who already own collectors, detectors and recorders. The Econo Gradient Pump • The Econo Gradient Pump is designed to capture the low, low end of the protein purification market. • The EGP is the heart of a modular chromatography system. • Customers may add fraction collectors, UV and conductivity monitors and chart recorders from Bio-Rad as needed (up-gradable). Programming Features • VERY SIMPLE PROGRAMMING! • 4 Modes – REMOTE (control from Duo-Flow) – SETUP – MANUAL – PROGRAM EGP faceplate controls Remote Mode • Control from Biologic Duo-Flow System running software version 3.0 – for use as a load pump – for post-column operations by selecting flow rate, %B and Split % Setup Mode • Used to select the “system” parameters; – Flow rate in ml/min or rpm – Flow rate calibration (tubing I.D. or collected volume) – Time or Volume units – Splitter Valve period – Revolution counter – LCD brightness – Choice of fraction collector – Erase program – Beeper ON or OFF Manual Mode • Manual mode controls three different parameters: – Flow rate (ml/min or rpm) – %B – Splitter Valve % split or Diverter Valve position (note that only one of these valves can be connected at the same time) Program Mode • Uses 6-steps or breakpoints – time or volume units – %B – flow rate – one collection window – fraction size programmed at the collector 1 2 3 4 5 6 Programming a gradient with breakpoints Time or Volume % B ml/min 0 0 1.0 5 0 1.0 25 100 1.0 35 100 1.0 45 0 1.0 50 0 1.0 Diverter valve programming One Collection Window (Time or Volume) may be programmed. From COLLECT To 8 min 23 min Splitter valve applications • New option for BioLogic system and HPLC customers who wish to split the column flow into another collector for analysis. • The “splitter period” defines the application – “continuous split” versus “sampling split”. Continuous stream splitting 95% to primary collector 5% to secondary collector Controlling the splitter valve for process sampling, e.g. from a fermenter Splitter valve used for process sampling 600 sec 1200 sec0 sec Sample 1 Sample 3Sample 2 Splitter valve is ON for 24 secs, OFF for 576 secs Peripheral Devices • Chart Recorder (Model 1327) • Gradient Monitor (Model EG-1) • Fraction Collector (Model 2110) • UV Monitor (Model EM-1) The BioLogic LP System LP specifications: Features and Benefits • A fully integrated low-pressure system • Dual-channel peristaltic pump i.e 25 psi, flow rate to 40 mL/min. • Cold-room compatible • Compact package with integral rack • Single-point control of all peripherals • Easy to maintain pump, wide choice of media • Essential for labile proteins • Fits into cold-boxes and cramped laboratories LP Valves: Features and Benefits • Controls Gradient Mixer valve • Controls Diverter Valve (optional) • Column bypass valve (optional). • Selection of 5 buffers (with SV-5 valve). • For reproducible & accurate gradients • Divert void volume to waste, permits collection windows • Unique. Purge the System without removing the column • Automatically load large samples, use extra buffers to clean/sanitize the column and add storage solution LP Software: Features and Benefits • 50-Method Storage and Naming • Up to 50-step programming in Time or Volume units • Edit method in progress • Up to 50 collection windows with Threshold • Delay volume feature • User convenience, easy to find any method • Programming flexibility • Easy to make changes without aborting the run • Advanced feature useful for efficient separation and fraction collection • Synchronizes fraction advance marks and fraction collection LP Software: Features and Benefits • 3 programmable Alarms • “Hold gradient %B” feature. • Multi-run feature • On-line Help feature • Uses Model 1327 chart recorder and/or LP Data View software • Reminders to switch valves, check status • Helps in developing methods • Automate separations • Benefits new users • LP Data View is a unique feature that replaces a chart recorder BioLogic LP Data View LP Data View features • LP Data View for real-time capture of chromatogram data on a PC – Windows 95, 98, and NT4.0 – Operates like a chart recorder with remote start/stop from the BioLogic LP – Scaling of the chromatogram during a run – Records the Method automatically at run end – Chromatogram annotation – View previous runs while collecting data Run screen Export Data Methods and Notes Columns and media for the EGP and BioLogic LP • Most media with a bead size greater than 25 micron. • All Bio-Rad’s MacroPrep line of Process media. 1. QuadTec UV/Vis Detector 2. SV5-4 Select Valve 3. BioLogic Maximizer Mixer 4. AVR9-8 Switching Valves 5. BioLogic Maximizer Buffer Blending System 6. pH Monitor 7. F40 Workstation 8. BioFrac Fraction Collector 9. AVR 7-3 Sample Inject Valve 10. UNO Q1 Column 11. BioLogic Rack 12. BioLogic DuoFlow Software 13. USB Bitbus Communicator 14. Dell Controller The Biologic Duo-Flow System Unlimited Growth Potential Up-Gradable System S = Standard U = Upgradable F10 Work staion F40 Work station BioFrac Fraction Collector QuadTec UV/Vis Detector Maximizer Valve System pH monitor 214nm kit BioLogic DuoFlow basic system 10 ml/min flow rate to 3,500 psi 254/280 nm detection S U U U U U U BioLogic DuoFlow standard system 10 ml/min flow rate to 3,500 psi 254/280 nm detection Fraction collection S U S U U U U BioLogic DuoFlow QuadTec basic system 10 ml/min flow rate to 3,500 psi Simultaneous 4- wavelength detection S U U S U U S BioLogic DuoFlow QuadTec standard system 10 ml/min flow rate to 3,500 psi Simultaneous 4-wavelength detection Fraction collection S U S S U U S BioLogic DuoFlow Maximizer 20 system 20 ml/min flow rate to 3,500 psi 254/280 nm detection Buffer blending pH monitoring Fraction collection S U S U S S U BioLogic DuoFlow Maximizer 80 system 80 ml/min flow rate to 1,000 psi 254/280 nm detection Buffer blending pH monitoring Fraction collection U S S U S S U Up-Gradable System BioLogic DuoFlow Pathfinder 20 system 20 ml/min flow rate to 3,500 psi Simultaneous 4-wavelength detection Buffer blending pH monitoring Fraction collection S U S S S S S BioLogic DuoFlow Pathfinder 80 system 80 ml/min flow rate to 1,000 psi Simultaneous 4-wavelength detection Buffer blending pH monitoring Fraction collection U S S S S S S Up-Gradable System Process Chromatography - To the for profit accounts Process Chromatography • Large market - $130M in 1997 • Rapidly growing @ 15% per year • HIGHLY profitable business Process Chromatography Market • Conservative, slow to make changes • A few large competitors - Pharmacia, Toso- Haas, Merck Market Requirements • Cost effective manufacturing process • Large resin batch sizes • Batch-to-batch consistency • Guaranteed resin supply • Ease of validation Chromatography Needs Characteristic Industrial R&D PD Manufacturing Capacity * *** *** Flow Rate ** *** *** Resolution *** ** ** Pressure * *** *** Cost ** * * Economics (Cost/gm) ** *** *** • Production of Biological Molecules – Vaccines – Large Molecule Therapeutics – Plasmids/Oligonucleotides – Blood Products – Blood Additives – Antibodies for Diagnostic Kits – Transgenic Proteins – Bulk Enzymes – Dietary Supplements – Milk proteins Applications Goal • To position Bio-Rad as a – Major supplier of supports for process chromatography – A supplier of a new generation of supports that solves the customers separation problems – Supplier Process columns (distribute Verdot columns) How To Find The Process Customer • Do you do much chromatography? – Does your company (have plans to) produce biomolecules on a large scale? – Do you have a process development/pilot scale group? – What size columns are you using? – Are you currently evaluating supports? Advantages of Macro-Prep for Process Chromatography • Enables improved cost effectiveness in manufacturing due to – High throughput/linear flow rates – Enhanced chemical stability – High binding capacities Benefits Bio-Rad provides as a chromatography supplier • Bio-Rad is a large, well established supplier providing – Regulatory support – Large resin batches – Resin batch-to-batch consistency – ISO 9001 manufacturing – Sales & technical support – A long term commitment to Process Chromatography business Conclusion • Disadvantage • Slow Sales Development – Year 1: Evaluate – Year 2-3: Process Development – Year 3-4-5: Clinical Trials – Years 3-25: Production • Advantage • Big Money – Long Stable Business – Very Little Maintenance once Process is running The Macro-Prep User • Industrial – R&D Scientists – Process Development Scientists – Chemical Engineers – Manufacturing Personnel • Academic – Research Scientists – Graduate Students Chúc các bạn học tốt