Small-scale purification and analysis of protein samples by size exclusion chromatography (SEC)

Transcription

1 Small-scale purification and analysis of protein samples by size exclusion chromatography (SEC) New generation, agarose-based columns - What s in it for you?

2 Content 1. Fundamentals of SEC 2. Introduction to the new generation, agarose-based SEC columns 3. New-generation SEC columns from GE vs predecessors 4. Application examples 5. Technical data 6. Tips and tricks for improved SEC results 7. Summary 2

3 Fundamentals of SEC

4 Principles of size exclusion chromatography Separates molecules based on size Characteristics Nonbinding technique Mild conditions Any buffer can be used Limited in sample volume By nature a slow technique Largest molecules elute first A well-packed SEC column is critical for high-resolution separations 4

5 Critical to get good results in SEC: well-packed columns High resolution is key For protein analysis applications: it is a way to obtain high-quality data For protein purification applications: it is a way to obtain highly pure proteins Shorter run time is beneficial SEC is a slow technique by nature. Reducing the run time is a way to Save time Increase throughput in the lab Superdex Increase and Superose Increase prepacked columns have been developed to address the need to maximize resolution and reduce the run time 5

6 SEC can be used for preparative purposes... SEC is usually used as a last polishing step to obtain pure target molecules in sufficient amount Affinity chromatography to isolate the target protein (AC) Size exclusion chromatography to remove remaining impurities or aggregates (SEC) Pure protein SDS-PAGE AC SEC MBPTrap and Superdex are trademarks of General Electric Company. 6

7 SEC is also used for various analytical purposes For powerful separation by size under native conditions Different proteins have different sizes. A protein can occur in different size forms (monomeric, aggregated, degraded, complexed). Different size forms exhibit different functions. Many protein size forms are held together by noncovalent means that are preserved under native conditions To check the quality of a protein preparation in order to understand whether the protein prep is fit for purpose: Qualify a protein prep for further use Monitoring process development, manufacturing, or formal QC for batch release To study protein properties in order to understand protein function and thereby characterize the protein: Evaluate protein stability, aggregation, and degradation Study protein interaction and complex formation Target protein Aggregated target protein Complexed target protein Degraded target protein Other proteins or contaminants 7

8 Size Exclusion Chromatography handbook available for download Download SEC handbook now 8

9 Introduction to the new generation, agarose-based SEC columns

10 GE offers prepacked SEC columns for user convenience and reproducible results Two product ranges depending on sample volumes Broad range of biomolecules that can be separated SEC columns Sample volume < 500 μl Sample volume < 13 ml Small-scale preparative purification and/or analysis Columns packed with Superose resins Superdex resins Preparative purification HiLoad Superdex pg HiPrep Sephacryl 10

11 GE offers prepacked SEC columns for user convenience and reproducible results Two product ranges depending on sample volumes Broad range of biomolecular sizes that can be separated SEC columns New generation resins, named Increase available for small-scale purification/analysis Sample volume < 500 μl Sample volume < 13 ml Small-scale preparative purification and/or analysis Columns packed with Superose resins Superdex resins Preparative purification HiLoad Superdex pg HiPrep Sephacryl 11

12 How to know which resin to choose? Superdex 30 Increase You have four choices of the new generation, agarose-based SEC resins for small-scale preparative purification and analysis This image is reproduced from Wikimedia Commons ( and is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. 12

13 Effect of the different fractionation ranges, as seen in separation of a mix of standard proteins. Superose 6 Increase: optimal resolution M r ~ Superdex 200 Increase: optimal resolution M r ~ Superdex 75 Increase: optimal resolution M r ~ Superdex 30 Increase (not in the Fig) is for peptides and other small biomolecules 13

14 Three column dimensions to fit different needs * * Not available as a standard product for Superdex 30 Increase, but can be ordered as custom-designed product 14

15 Explore Superdex Increase and Superose Increase columns Explore columns 15

16 New-generation SEC columns from GE vs predecessors: impact on resolution and speed

17 GE is renewing legacy Superose and Superdex columns with new-generation SEC Increase columns Legacy columns (and last date of sales) New-generation SEC columns Superdex 200 (Dec 2015) Superose 6 (Dec 2016) Superdex Peptide (Dec 2017) Superdex 75 (Dec 2018) Superose 12 (Dec 2018) Superdex 200 Increase Superose 6 Increase Superdex 30 Increase Superdex 75 Increase Any of the above suitable for your target sample For more information, please contact your local GE representative 17

18 Increase = improved performance vs predecessors Higher resolution Example: Superdex 200 Increase delivers up to 60% higher resolution than Superdex 200 at the same flow rate Quicker runs Example: Superdex 75 Increase reduces run time by two-thirds compared with Superdex 75 with maintained resolution Superdex /300 GL Superdex 200 Increase 10/300 GL Superdex 75 10/300 GL Run time 60 min Superdex 75 Increase 10/300 GL Run time 16 min Flow rate 0.4 ml/min Flow rate 1.5 ml/min Flow rate 0.5 ml/min Run time 48 min 18

19 Superdex 30 Increase vs Superdex Peptide Up to 50% higher resolution on Superdex 30 Increase Run time reduced by two-thirds with maintained resolution on Superdex 30 Increase Superdex Peptide 10/300 GL Superdex 30 Increase 10/300 GL Superdex Peptide 10/300 GL Superdex 30 Increase 10/300 GL mau DAD1 B, Sig=214,4 Ref=off (Flash\Ninni \ D) mau DAD1 B, Sig=214,4 Ref=off (Flash\Ninni \ D) Sample mix M r 1. Cytochrome C Aprotinin Gastrin Glycine Glycine Glycine 75 mau DAD1 B, Sig=214,4 Ref=off (Flash\Ninni \ D) mau DAD1 B, Sig=214,4 Ref=off (Flash\Ninni \ D) min Flow rate: 0.5 ml/min min min (0.4 ml/min) min min (1.2 ml/min) min 19

20 Superdex 75 Increase vs Superdex 75 Up to 50% higher resolution on Superdex 75 Increase Reduced run time by two-thirds with maintained resolution on Superdex 75 Increase Flow rate 0.8 ml/min Run time 30 min Flow rate 0.4 ml/min Run time 60 min Flow rate 1.5 ml/min Run time 16 min Sample mix: M r 1. Conalbumin Ovalbumin Carbonic anhydrase Cytochrome C Aprotinin Vitamin B Superdex 75 10/300 GL Superdex 75 Increase 10/300 GL Superdex 75 10/300 GL Superdex 75 Increase 10/300 GL 20

21 Superdex 200 Increase vs Superdex 200 Up to 60% higher resolution on Superdex 200 Increase Reduced run time by two-thirds with maintained resolution on Superdex 200 Increase Flow rate 0.5 ml/min Run time 48 min Flow rate 0.5 ml/min Run time 48 min Flow rate 1.5 ml/min Run time 16 min Sample mix: M r Thyroglobulin Ferritin Aldolase Conalbumin Ovalbumin Carbonic anhydrase Aprotinin Superdex /300 GL Superdex 200 Increase 10/300 GL Superdex /300 GL Superdex 200 Increase 10/300 GL 21

22 Superose 6 Increase vs Superose 6 Up to 40% higher resolution on Superose 6 Increase Twice as fast with maintained resolution on Superose 6 Increase Flow rate 0.5 ml/min Run time 48 min Flow rate 0.5 ml/min Run time 48 min Flow rate 1.0 ml/min Run time 24 min Sup PR1 001:10_UV1_280nm Sup PR1 001:10_Inject Sup6Inc PR1 B 001:10_UV1_280nm Sup6Inc PR1 B 001:10_Inject mau mau Standard proteins: M r Thyroglobulin Ferritin Aldolase Ovalbumin Ribonuclease A Aprotinin ml ml Superose 6 10/300 GL Superose 6 Increase 10/300 GL Superose 6 10/300 GL Superose 6 Increase 10/300 GL 22

23 Improved detection of both antibody aggregates and fragments using Superdex 200 Increase Superdex /300 GL Superdex 200 Increase 10/300 GL mau SDX200 old Mab5 001:10_UV1_280nm SDX200 old Mab5 001:10_Inject mau SDX200 old Mab5 001:10_UV1_280nm SDX200 old Mab5 001:10_Inject Sample: Mab X mau Mab5 nr 2 001:10_UV1_280nm Mab5 nr 2 001:10_Inject mau Mab5 nr 2 001:10_UV1_280nm Mab5 nr 2 001:10_Inject R s R s ml ml min min 23

24 High lot-to-lot consistency for Superdex 200 Increase columns and resins Column-to-column reproducibility Batch-to-batch reproducibility 1 2 Columns: Superdex 200 Increase 10/300 GL Sample volume: 50 µl (0.2 % CV) Buffer: 20 mm NaH 2 PO 4 ph 7.4, 300 mm NaCl Flow rate: 0.75 ml/min Detection: 280 nm Resolution Retention volume 3 Overlay of three different 10/300 columns packed with the same Superdex 200 Increase resin lot: Peak 1 mab monomer (M r ) Peak 2 Fab (M r ) Peak 3 dab (M r ) Comparison of six different Superdex 200 Increase resin batches (same sample as to the left): Resolution: minor differences (RSD < 6%) Retention volume (ml) : minor differences (RSD < 10%) RSD = relative standard deviation 24

25 Rigid, small beads with a narrow particle size distribution and narrow specifications make the difference Superdex 30 Increase Superdex 75 Increase Superose 6 Increase Superdex 200 Increase Superdex Peptide Superdex 30 Increase Superdex 75 Superdex 75 Increase Superdex 200 Superdex 200 Increase Superose 6 Superose 6 Increase Cross-linked agarose and dextran matrix Cross-linked agarose and dextran matrix Cross-linked agarose and dextran matrix Cross-linked agarose matrix M r ~ fractionation range M r ~ fractionation range M r ~ fractionation range M r ~ fractionation range Regular pressure/flow properties Rigid beads with high pressure/flow resistance Regular pressure/flow properties Rigid beads with high pressure/flow resistance Regular pressure/flow properties Rigid beads with high pressure/flow resistance Regular pressure/flow properties Rigid beads with high pressure/flow resistance 13 µm average bead size 9 µm average bead size 13 µm average bead size 9 µm average bead size 13 µm average bead size 8.6 µm average bead size 13 µm average bead size 8.6 µm average bead size ph stability: Operational 1 : ph 3 12 Cleaning in place 2 : ph 1 14 ph stability: Operational 1 : ph 3 12 Cleaning in place 2 : ph 1 14 ph stability: Operational 1 : ph 3 12 Cleaning in place 2 : ph 1 14 ph stability: Operational 1 : ph 3 12 Cleaning in place 2 : ph 1 14 Benefits of ph-stable agarose-based SEC resins: Prolonged column lifetime Minimized carry-over between runs Possible use of basic buffers 1 ph range where resin can be operated without significant change in function. 2 ph range where resin can be subjected to cleaning- or sanitization-in-place without significant change in function. 25

26 Superose 12 is to be replaced by a choice of four new-generation SEC columns Superose 12 Superdex 30 Increase Superdex 75 Increase Superdex 200 Increase Superose 6 Increase 26

27 Choosing SEC Increase column for different sized biomolecules used on Superose 12 Same sample mix run on Superose 12 and all SEC Increase columns Use column depending on molecular weight of target protein Standard Protein Molecular weight (Mr) Superdex 30 Increase Superdex 75 Increase Superdex 200 Increase Superose 6 Increase > Ferritin ConAlbumiin Ovalbumin Carbonic Anhudrase RNAse Aprotinin Vitamin B Cytidine 240 > 100 For some size ranges there are several resins to choose from. Make the choice that you think will fit best with your specific target molecules using the table above as a guide. 27

28 Data files available for download (click on the document of interest) Superdex 30 Increase >> Superdex 75 Increase >> Superose 6 Increase >> Superdex 200 Increase >> DOWNLOAD DOWNLOAD DOWNLOAD DOWNLOAD 28

29 Application examples

30 mab aggregate analysis with Superdex 200 Increase 10/300 GL migg1 15 ug_ 0105_ CH2 3 A. B Intensity [µv] Details: Column: Superdex 200 Increase 10/300 GL Sample: Mouse IgG 1, 1.0 mg/ml Sample volume: 15 µl Flow rate: 0.6 ml/min Buffer: 100 mm phosphate, 200 mm NaCl, ph 6.8 System: HPLC system Retention Time [min] Analysis of monoclonal mouse IgG 1 antibody aggregates Retention Time [min] Superdex 200 Increase delivers high-resolution analysis of dimers and higher aggregates 30

31 mab aggregate screening with Superdex 200 Increase 5/150 GL Run time 4 min Detection limit 0.14% aggregates Superdex 200 Increase delivers quick detection of low levels of aggregates 31

32 Rapid detergent screening for crystallization of membrane proteins using Superdex 200 Increase 5/150 GL A recombinant membrane protein (CE07) was solubilized in four different detergents and the column was equilibrated with buffer containing the same detergent. The screening was performed to evaluate the best detergent for further crystallization. Superdex 200 Increase short columns enables analysis time down to 6 min We thank Dr. Per Moberg at the Karolinska Institute, Stockholm, Sweden for collaboration and for providing data on this detergent screening. 32

33 Purification of histidine-tagged protein with Superdex 75 Increase Using Superdex 75 Increase 10/300 GL, 5 mg of a size homogenous target protein was successfully purified in a single run (A) (B) (C) Purification of a partially purified size-heterogeneous histidinetagged (his-tagged) protein on Superdex 75 Increase 10/300 GL Analysis of the final pool on Superdex 75 Increase 5/150 GL SDS-PAGE Sample volume: 500 µl Flow rate: 0.2 ml/min 1. Molecular weight marker 2. Blank 3. Pool (fractions 3 to 12) from A 4. Pool (fractions 13 to 18) from A 5. Start material 6. Blank 7. Molecular weight marker 33

34 SEC analysis reveals size-heterogeneity not seen is SDS-PAGE, since SDS dissociates noncovalent dimers and oligomers This can be seen when comparing lane no 4 on the SDS- PAGE gel, where it seems to be mainly one large single band, but in the SEC chromatogram B you can see two larger aggregates on the left-hand side of the main peak. 1. Molecular weight marker 2. Blank 3. Pool (fractions 3 to 12) from A 4. Pool (fractions 13 to 18) from A 5. Start material 6. Blank 7. Molecular weight marker 34

35 Studying aggregation and degradation over time using Superdex 75 Increase 5/150 GL Aggregation protein 1 Degradation protein 2 A A ,5 1,7 1,9 ml Running conditions: Column: Superdex 75 Increase 5/150 GL Sample volume: 10 μl Flow rate: 0.5 ml/min System: HPLC with autosampler Abs A ,7 2,2 2,7 ml week 2 week 4 week week 2 week 4 week 0 0 0,5 1 1,5 2 2,5 3 3,5 Retention volume (ml) 0 0 0,5 1 1,5 2 2,5 3 3,5 Retention volume (ml) Superdex 75 Increase 5/150 GL was used to monitor small changes in size homogeneity of two proteins stored under different conditions and over different time periods (weeks). The HPLC system used was equipped with an autosampler to allow analysis of many samples during long, unattended, overnight SEC runs. 35

36 Purification of oligomerized pathogenic protein with Superose 6 Increase 3.2/300 (A) Monomeric α-synuclein (B) After incubation with aldehyde, monomeric α-synuclein has been oligomerized and no monomeric form can be detected Superose 6 Increase 3.2/300 was used for preparation of oligomerized α-synuclein for further in vitro and in vivo studies We thank Dr. Joakim Bergström, Rudbeck Laboratory, Uppsala University, Sweden for collaboration and for providing this data. 36

37 Purification of a membrane protein complex with Superose 6 Increase 5/150 GL Column: Superose 6 Increase 5/150 GL Sample: ATP synthetase complex from E. coli membrane Sample volume: 25 µl Buffer: 20 mm Tris-HCl, 150 mm NaCl, 10% glycerol, 250 µm MgCl 2, 0.05% dodecyl maltoside, ph 8.0 Flow rate: 0.15 ml/min System: ÄKTAmicro in cold room Partially purified ATP synthetase complex was further purified on Superose 6 Increase 5/150 GL to remove remaining contaminants Run time: 16 min Purified complex was used for subsequent structural and molecular mechanism studies. 37

38 Estimation of molecular weight distribution of a protein hydrolysate with Superdex 30 Increase 10/300 GL Improved resolution with Superdex 30 Increase Response [mau] Response [mau] x x Superdex 30 Increase * * * * Retention time [min] Superdex Peptide * * * * * Retention time [min] Columns: Superdex 30 Increase 10/300 GL Superdex Peptide 10/300 GL Sample: Tryptic hydrolysate from lentils Sample volume: 50 µl Buffer: 20 mm phosphate buffer, 280 mm NaCl, ph 7.4 Flow rate: 0.75 ml/min System: HPLC * * There is a growing interest in protein hydrolysates from inexpensive sources. Short bioactive peptides can have higher nutritive value, for example, as antioxidants. In the characterization of protein hydrolysates, it is important to analyze the size distribution. Here is a comparison of Superdex Peptide and Superdex 30 Increase columns. The results obtained on Superdex 30 Increase showed improved resolution of a lentil hydrolysate compared to Superdex Peptide. 38

39 Impurity analysis of different lots of (Ile 7 )-Angiotensin III on Superdex 30 Increase 10/300 GL Superdex 30 Increase 10/300 GL was used for impurity analysis of two lots of a peptide: Impurity Lot A Lot B Main Impurity (Peak 1) 1.2% (RSD 0.45) 0.1% (RSD 1.72) Total 3.2% (RSD 0.30) 0.7% (RSD 1.62) Column: Superdex 30 Increase 10/300 GL Sample: Different lots of (Ile 7 )-Angiotensin III Sample volume: 100 µl Buffer: 20 mm phosphate buffer, 280 mm NaCl, ph 7.4 Flow rate: 0.5 ml/min System: HPLC Analytical SEC offers quantitative assessment of size inhomogeneities of active peptide in native conditions. 39

40 Application notes available for download Analysis of conformational changes of a membrane protein using size exclusion chromatography DOWNLOAD Continuous chromatography in downstream processing of a monoclonal antibody DOWNLOAD Size exclusion chromatography analysis of papain-cleaved monoclonal antibody using Superdex 200 Increase columns DOWNLOAD Three-step monoclonal antibody purification processes using modern chromatography resins DOWNLOAD 40

41 Tips and tricks for improved SEC results

42 Minimize internal system volumes to optimize resolution Examples of LC systems compatible with Superdex Increase and Superose Increase columns ÄKTA pure 25 ÄKTApurifier 10 1 HPLC system Large internal system volumes in a liquid chromatography system can negatively affect the resolution obtained with SEC Increase columns The system and its configuration affect resolution Improve the resolution by using: Short and narrow tubing Low component internal volume 1 ÄKTApurifier chromatography systems are now discontinued and replaced with ÄKTA pure. Discover ÄKTA pure at 42

43 Both sample volume and flow rate affect the resolution in an SEC run See the chromatograms to the left for comparison data Rule of thumb for sample volume: 0.5% of bed volume for optimal resolution in analytical runs Up to 2% of bed volume for preparative runs Lower flow rates deliver higher resolution 43

44 Perform regular cleaning-in-place to keep the column in good shape Sodium hydroxide is a very efficient cleaning solution and it is possible to use with these agarose-based SEC resins. In all instructions for use, a protocol for performing cleaning of the column is at hand. Recommendations: Regular cleaning after 10 to 20 separation cycles Initial functional test as reference for performance follow up 44

45 Additional tips in our Protein Purification troubleshooting guide Download the guide 45

46 Summary

47 Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, and Superose 6 Increase columns are new-generation SEC columns for high-resolution separations under mild conditions for proteins and other biomolecules offer higher resolution and shorter run times than their predecessors Molecular weight ranges (M r ) Superose 6 Increase: ~ 5000 to ~ Superdex 200 Increase: ~ to ~ Superdex 75 Increase: ~ to ~ Superdex 30 Increase: ~ 100 to ~

48 Useful tools and ordering information Size exclusion chromatography handbook gelifesciences.com/proteinhandbooks New-generation SEC columns Product code Superdex 200 Increase 10/300 GL Superdex 200 Increase 5/150 GL Superdex 200 Increase 3.2/ Purify app gelifesciences.com/purify More information on our new-generation SEC columns: Superose 6 Increase 10/300 GL Superose 6 Increase 5/150 GL Superose 6 Increase 3.2/ Superdex 75 Increase 10/300 GL Superdex 75 Increase 5/150 GL Superdex 75 Increase 3.2/ Superdex 30 Increase 10/300 GL Superdex 30 Increase 3.2/

49 gelifesciences.com GE, the GE Monogram, ÄKTA, HiLoad, HiPrep, Sephacryl, Superdex, Superose, and Tricorn are trademarks of General Electric Company. 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 General Electric Company. GE Healthcare Bio-Sciences AB Björkgatan Uppsala SwedenE-7 Uppsala Sweden 49

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