Introduction to Size Exclusion Chromatography (SEC)

Transcription

1 Introduction to Size Exclusion Chromatography (SEC) Sumalee Phumratanaprapin Channel Account Manager for BP, Indochina. Imagination at work AA July 2015

2 Content What is size exclusion chromatography (SEC) What is SEC used for? Principles Experimental set up Tips and hints AA July 2015

3 What is size exclusion chromatography (SEC)

4 What is SEC? Separation by size and shape Non-binding One buffer Simple to do and understand Sample limitation AA July 2015

5 What is SEC used for?

6 Main usage of SEC 1) Group separation Group separation: Desalting buffer exchange Adjusting ph, buffer type, salt concentration during sample preparation, e.g. before an assay Removing interfering small molecules, e.g. EDTA, Urea Removing small reagent molecules, e.g. fluorescent labels, radioactive markers AA July 2015

7 Main usage of SEC 2) High resolution fractionation High resolution fractionation Excellent during the polishing stage Removes dimers and other aggregates Transfers protein to buffer solution ready for the next set of experiments AA July 2015

8 Main usage of SEC 3) Characterization Characterization Size homogeneity analysis Estimating molecular size Rapid purity check AA July 2015

9 Why choose SEC? Advantages Separates by size (and shape) Fast method for buffer exchange Very gentle, high yields Simple Disadvantages Limited sample volume Dilute the sample Preparative gel filtration is quite slow technique Works in majority of buffer solutions Removes dimers and aggregates AA July 2015

10 Application examples for desalting buffer exchange Disposable PD-10 Desalting columns HiTrap Desalting HiPrep 26/10 Desalting albumin NaCl ml sec AA July 2015

11 Application examples for high resolution fractionation Purification of recombinant IGF-1 Purification of recombinant phosphatase AU IGF-1 Column: Sample: 1 2 time h HiLoad 16/600 Superdex 75 pg IGF-1, ZZ fusion protein and uncleaved material Buffer: 0.15 ammonium acetate, ph 6.0 Flow rate: 0.75 ml/min 22.5 cm/h Column: Sample: Buffer: Flow rate: HiLoad 16/600 Superdex 75 pg 4 ml concentrated eluate from a HIC run containing rphosphatase 25 mm Tris-HCl, 0.3 M sodium chloride, 1 mm EDTA, 2mM DTT, ph ml/min 15 cm/h AA July 2015

12 Application example for characterization Size homogeneity analysis mau dimer monomer Column: Sample: Superdex 75 10/300 GL reccys-prot., protein solution containing a mixture of dimers and monomers Sample volume: 200 µl Elution buffer: 50 mm TrisHCl, 1 mm EDTA, 0.15 M NaCl, ph 8.4 Flow rate: 0.5 ml/min System: Detection: ÄKTA System A 280 nm ml AA July 2015

13 Characterization Estimation of molecular size Calibration curve manually or automatically V R log M r Plotting the elution volume versus the logarithm of molecular mass yields a calibration curve. Molecular size table dialog box in the UNICORN software Analysis Module (add on option) AA July 2015

14 Rapid purity check and screening Superdex 75 10/300 GL Column volume 24 ml Flow rate 0.6 ml/min Sample volume 100 µl Analysis time 47 min Superdex 75 5/150 GL Column volume 3 ml Flow rate 0.3 ml/min Sample volume 12 µl Analysis time 12 min Superdex :10_UV1_280nm Superdex :10_Inject mau min Superdex 75 nr 17001:10_UV1_280nm Superdex 75 nr 17001:10_Inject mau min For rapid analysis and homogeneity test in screening experiments - 4 times faster than long SEC column Excellent choice when buffer consumption is more important than highest resolution - 8 times less volume than long SEC column Best choice when sample volume is limited min 12 min! AA July 2015

15 Principles

16 Matrix structure of SEC chromatography media Agarose A good matrix for SEC contains about 95% water Hypothetical structure for Superdex dextran cross-linked agarose cross-section from Superdex particle AA July 2015

17 How does SEC work? Spherical particles packed into a column. 2. Sample applied. 3. Buffer mobile phase and sample move through column, molecules diffuse in and out of matrix. 4. Large molecules leave the column first followed by smaller molecules in order of size, molecules larger than the matrix pores pass straight through. Molecules elute in order of size. The largest molecules elutes first; the smaller ones elute last AA July 2015

18 The number of peaks is limited compared to other techniques Maximum number of peaks in RPC >150 Maximum number of peaks in SEC ~ ml Remember: we are not purifying peaks, but proteins and peptides! AA July 2015

19 Experimental set up

20 How to plan your SEC purification? Answer to three essential questions 1. Is it a group separation or high resolution fractionation? 2. What is the molecular weight of the target protein and contaminants? 3. What is the sample volume? AA July 2015

21 Question 1. Is it a group separation or high resolution fractionation? What chromatography media to use? Desalting/Buffer exchange Sephadex High resolution fractionation Superdex, Superose, Sephacryl A well-packed column is essential use a pre-packed column! AA July 2015

22 Question 2. What is the molecular weight of the target protein and contaminants? Choose a medium where the molecular weight of your target protein is in the middle of the fractionation range AA July 2015

23 Question 3. What is the sample volume? Desalting Product HiTrap Desalting * (5 ml) HiPrep Desalting * (53 ml) Max sample volume 1.5 ml 15 ml * Possible to connect several columns to increase sample volume Fractionation Column dimensions Analytical scale (µl)** Max sample volume 10/ µl 5/ µl Small volume buffer exchange Product Format Max sample volume PD-10 desalting columns PD MidiTrap G-25 PD MiniTrap G-25 Gravity column Gravity column Gravity column 2.5 ml 1 ml 0.5 ml PD SpinTrap G-25 Spincolumn 130 µl PD MultiTrap G well filter plate 130 µl 3.2/30 25 µl Preparative scale (ml)*** 16/60 5 ml 26/60 13 ml **Tricorn and Precision columns ***HiLoad and HiPrep columns Anything larger, purchase media, empty column and pack yourself AA July 2015

24 Peak width depends on particle size Superdex Peptide µm Superdex 30 prep grade µm AU AU retention time min retention time min AA July 2015

25 Column size depends on sample volume Desalting/Buffer exchange Sample volume up to 30 % of the total column volume can be applied Length of the column is not so important High resolution fractionation Sample volume of 0.5 % to 4 % of the total column volume can be applied Column length required: cm For rapid purity check, 15 cm length can be good enough AA July 2015

26 Resolution depends on column length 2 x Superdex Peptide column 10 x 300 mm i.d x bed height AU x Superdex Peptide column 10 x 300 mm i.d x bed height AU retention time min retention time min AA July 2015

27 Resolution depends on sample volume Superdex Peptide 10 x 300 mm column A µl Retention volume (ml) AU µl Retention volume (ml) AU µl Retention volume (ml) AA July 2015

28 High resolution SEC columns Schematic overview of resolution and sample volume for pre-packed high resolution SEC columns TM TM TM TM TM AA July 2015

29 Tips and hints

30 Tips and hints Maintenance of your SEC column is important to keep it in good shape Check the column efficiency regularly If air enters the column, run buffer through the column at slow flow rate Sample preparation Always filter or spin the sample to remove particles before applying onto the column For better resolution Reduce the sample volume Reduce the flow rate Change to a medium with smaller beads Connect two columns in series Check system dead volumes and minimize them AA July 2015

31 Introduction to multimodal chromatography

32 Multimodal chromatography basics Multimodal: Having more than one mode of action Multiple interactions are intentionally used Earlier called mixed mode chromatography Useful when other chromatography techniques do not give the desired selectivity AA July 2015

33 Introducing multimodal chromatography Traditional resin Ion exchange SO 3 - Hydrophobic interaction Multimodal resin Modulates the action of traditional resins Generates new selectivities Affinity AA July 2015

34 Principles of multimodal chromatography

35 Multimodal ion exchange resins Multimodal cation exchanger Capto TM MMC NH Multimodal anion exchanger Capto adhere Potential for ionic interactions OH OH O O N + OH AA July 2015

36 Multimodal ion exchange resins Multimodal cation exchanger Capto TM MMC NH Multimodal anion exchanger Capto adhere Potential for hydrophobic interactions OH OH O O N + OH AA July 2015

37 Multimodal ion exchange resins Multimodal cation exchanger Capto TM MMC NH Multimodal anion exchanger Capto adhere Potential for hydrogen bonding OH OH O O N + OH AA July 2015

38 Binding strength Interaction mode, IEX or HIC? Concept Example: Antibody on Capto MMC NaCl (M) AA July 2015

39 When to use multimodal ion exchanger? When other chromatography techniques do not give the desired selectivity If salt tolerance is desired When an existing multi-step chromatography procedure needs to be shortened AA July 2015

40 Salt-tolerant medium Retain high binding capacity at high conductivity Feed material can be loaded without the need for dilution or buffer exchange AA July 2015

41 Highly selective removal of antibody aggregates in flow through mode Column: Capto TM adhere, 0.6 ml Sample: 160 mg purified monoclonal antibody (mab) with 6% aggregates Binding: 20 mm citrate, 300 mm NaCl, ph 6.5 Elution: 0.1 M acetic acid, ph 3.0 mab monomer mab aggregates AA July 2015

42 How to use multimodal chromatography?

43 Protocol development - considerations Technique Traditional ion exchange chromatography Hydrophobic interaction chromatography Low conductivity Binding No binding High conductivity No binding Binding Multimodal ion exchange chromatography?? AA July 2015

44 Protocol development - considerations A 280 Salt and ph elution on Capto MMC AA July 2015

45 Summary

46 When to use multimodal ion exchangers? When other chromatography techniques do not give the desired selectivity If salt tolerance is desired When an existing multi-step chromatography procedure needs to be shortened AA July 2015

47 How to use multimodal ion exchangers? Screen ph and conductivity for optimal running conditions Use Design of Experiments (DoE) Parallel screening formats can speed up the process Choose appropriate chromatography media: - Capto MMC and Capto adhere for excellent flow property - Capto MMC ImpRes and Capto adhere ImpRes for best resolution AA July 2015

48 Introduction Capto Core is designed for purification of viruses and other large biomolecules...is optimized for intermediate purification and polishing steps...is developed for flowthrough applications...is based on core bead technology...is a high productivity alternative to gel filtration in group separation Capto Core 700 offers: High purity and high yield High loading and short residence times Wide window of operation allows easy process design High productivity and improved process economy * * compared to gel filtration

49 Capto Core 700 concept

50 Concept Layered beads More dimensions to each bead SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 SO - 3 Layer 1 Layer 2 Layer 3 Design variables -Particle size -Pore size -Type of ligand -Ligand density Additional design variables -Number of layers -Layer thickness -Porosity modification -Type of ligand

51 Concept Core Beads Ligand exclusively in the core of the bead Pore size defines molecular weight cutoff Target in the core Impurity outside Bind-elute Target outside Impurity inside Flowthrough

52 Capto Core 700 Concept Capto Core µm inactive shell Viral particle N + Octylamine - Multimodal ligand Very strong protein binding Host cell proteins (HCP) DNA fragments Endotoxins Detergents Benzonase Etc 85 µm particle size

53 Features and benefits

54 Features and benefits High purity and yield with high load Purity and yield Capto Core 700 achieves the same HCP removal and virus recovery as gel filtration Load and dilution Capto Core 700 allows this purity and yield to be obtained with significantly higher load and without any dilution to the feed High productivity without compromising purity or yield!

55 Features and benefits Robust performance Ligand The multimodal octylamine ligand is both hydrophobic and positvely charged. This ligand allows for strong binding to a variety of contaminants. The octylamine ligand is enabled for use due to the core bead format of Capto Core 700. Robust performance The octylamine ligand will maintain its performance over a wide window of buffer conditions. Easy optmization and flexible process design!

56 Features and benefits Group separation and binding chromatography in one bead Cutoff The size of pores in the shell of Capto Core 700 defines the cutoff for which biomolecules can enter the core of the bead. Capacity Octylamine ligands secure strong binding to most contaminants that enter the core of the bead. High load, robust performance, and high purity and yield are ensured. Double the functionality, triple the benefits!

57 Features and benefits Wide window of operation Load High load enabled by the binding chromatography functionality of Capto Core 700. Flow rate The rigid high-flow agarose matrix in combination with core bead technology allows for high flow rates and short residence times. High productivity and flexible process design!

58 Features and benefits Process economy improvement Capto Core 700 vs gel filtration Gel filtration (size exclusion chromatography) Is a chromatographic technique often used in vaccine processes. Gel filtration offers the required purity and yield but gives relatively low productivity. Capto Core 700 Up to 100-fold higher load - the level of impurity determines load Up to 10-fold higher flow rates - short residence times and rigid base matrix Straightforward process design wide window of operation Easy scale-up linear scalaability and small footprints Compared with gel filtration, Capto Core 700 enables significantly improved productivity and greatly improved process economy without sacrificing purity or yield. Lower cost for scale-up low volumes required enables use of smaller equipment Will require CIP between cycles

59 Applications

60 Application Typical viral vaccine process Fermentation Harvest Clarification Purification Capto Core 700 Designed for intermediate purification to polishing steps in group-separation applications. For use in manufacturing of vaccines or purification of large biomolecules Formulation

61 Application Cell-based influenza vaccine Fermentation Harvest Fermentation A/H1N1/ Solomon Island Influenza virus strain (LAIV) MDCK Cells Cytodex 3 Clarification Clarification ULTA Prime GF 2.0 µm µm normal-flow filter capsule Purification Purification 1. Capto DeVirS 2. Capto Core 700 Formulation

62 Application Cell-based influenza vaccine Capto DeVirS Capto Core 700 STEP Titer (TCID 50 /ml) Virus HA Recovery (%) DNA removal (log red.) DNA/HA (ng/µg) HCP Removal (%) HCP/HA (µg/µg) Microfiltration Capto DeVirS Capto Core

63 Application Cell-based influenza vaccine Conclusions Excellent HA recovery over chromatography steps Excellent protein removal in process Virus infectivity not affected STEP Titer (TCID 50 /ml) Virus HA Recovery (%) DNA removal (log red.) DNA/HA (ng/µg) HCP Removal (%) HCP/HA (µg/µg) Microfiltration Capto DeVirS Capto Core

64 Application Egg-based influenza vaccine Process Ovalbumin reduction over Capto Core 700 Conclusions HA recovery over Capto Core 700 step > 90% Excellent ovalbumin reduction

65 Application IgM purification Conclusions > 95% HCP removal at > 75% recovery Optimized purity and recovery Possible to use Capto Core 700 for IgM purification When target close to cutoff keep residence time low!

66 Application Viral vaccines All marketed human viral vaccines are possible targets Virus and approximate size [nm] All large molecules that do not enter the Capto Core 700 beads can be purified!

67 Application Other potential applications Endotoxin removal Plasmid DNA...many others Purity: 80% to 90% removal of endotoxin Capacity: ~1.25 million EU/ml medium Removal of RNA, HCP, and endotoxins from plasmid DNA Benzonase removal Removal of phenol red from cell culture Sample prep applications Viral vectors for gene therapy Membrane proteins Vesicle / micelle purification Batch-mode applications...

68 Application Important during evaluation 1. Screen load volumes Measure breakthrough capacity by monitoring contaminant levels in collected fractions Larger contaminants i.e., gdna needs to be fragmented or removed by other technique Our experience: High load High yield High productivity 2. Optimize residence time Molecular weight close to cutoff - Often trade-off purity/recovery Short residence time higher recovery Long residence time higher purity 3. Optionally fine-tune your buffer conditions Wide window of operation (ph and conductivity) Citrate buffer, for example, is not compatible with ligand

69 Summary

70 Summary Positioning in processes Fermentation Harvest Clarification Purification Capto Core 700 Designed for intermediate purification to polishing steps in group-separation applications in manufacturing of viruses or other large biomolecules Most processes will require a combination of different chromatographic and/or other separation techniques together with Capto Core 700 to meet purity and regulatory demands Formulation Capto Core 700 will secure high purity and yield as well as high productivity and scalability

71 Need help to find the right product? Use the Purify App! This guide will help you to quickly select the right chromatography products for your protein separations. Available as a web version as well as for iphone, ipad, and Android AA July

72 Protein purification handbooks from GE Healthcare AA July 2015 GE Healthcare handbooks

73 Protein purification handbooks from GE Healthcare AA July 2015 GE Healthcare handbooks

74 Protein purification handbooks from GE Healthcare AA July 2015 GE Healthcare handbooks

75 GE, imagination at work, and GE monogram are trademarks of General Electric Company. HiLoad, HiPrep, HiTrap, MidiTrap, MiniTrap, MultiTrap, Sephacryl, Sephadex, SpinTrap, Superdex, Superose, Tricorn, UNICORN and ÄKTA are trademarks of General Electric Company or one of its subsidiaries. Android is a trademark of Google Inc. iphone and ipad are trademarks of Apple Inc 2015 General Electric Company All rights reserved. First published JUL All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire, HP7 9NA UK AA July

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