Gel Filtration (GF) Size Exclusion Chromatography (SEC)

Transcription

1 Gel Filtration (GF) Size Exclusion Chromatography (SEC) 1

2 Gel Filtration chromatography (GF) Principles of GF Fractionation range Parameters for resolution optimization Use of GF: MW/oligomeric state purification buffer exchange - QC Examples - Troubleshooting SEC-MALS 2

3 What is gel filtration? SEC is the most powerful chromatography technique for obtaining reliable information about the size of biomolecules under native conditions Separates molecules according to their hydrodynamic ratio (size, conformation & oligomeric state) Different fractionation ranges: beads with pores of welldefined sizes Mobile phase: almost all kind of buffers 3

4 Gel structure Agarose Dextran Void volume V o Volume of the gel matrix V s A hypothetical structure for Superdex Pore volume V i Different columns with beads of defined porosity: fractionation range The degree of cross-linking determines the size of the pores and therefore the fractionation range of the resin SEC is not an adsorption technique (unlike all other chromatographic procedures). 4

5 How does it work?

6 Terms and explanations V o = Void volume: volume of the solution outside the beads, or elution from very large molecules V e = the volume from the time the protein is placed until it appears in the effluent V i = volume of the solution inside the beads = V c - V s - V o V c = Total (geometric) volume of the column Vt = Elution volume for very small molecules Void volume V o V e Volume of the gel matrix V s V o V t Pore volume V i V c 6

7 Steric exclusion Gel bead Molecules are excluded from the gel bead to different extents according to their sizes. Largest molecules - excluded from pores, travel with the mobile phase, elute rapidly from column The volume at which large molecules elute is called the void volume, Vo (same as the volume of solution that surrounds the beads) 7 Intermediate size molecules spend different amounts of time both inside and outside the beads (partition between the mobile and stationary phase) The volume at which intermed.molecules elute is called the elution volume (Ve) and depends on the partition of the molecule between the Vo and Vs which is proportional to the distribution coefficient (K) Ve = Vo + KVs Smallest molecules enter the pores of the beads, are included in the matrix and retarded in their movement, spend most of the time in the stationary phase, elute last The volume at which small molecules elute corresponds to Vt (total volume of solution surrounding (Vo) and inside the beads, Vs) Vt = Vo + Vs

8 Column calibration: MW extrapolation of K av 1 Kav unknown molecule Ve V V t V o o Run MW standards and determine the elution volume for each (globular proteins) Calculate K av values Plot log (M r ) for each standard against the calculated K av 0 log (M r ) Selectivity curve is usually moderately straight over the range Kav=0.1 to Kav=0.7 8 Extrapolate MW of globular proteins according to Ve Limitations: protein is not globular; interaction with the resin

9 Shape effects K av Native proteins Denatured proteins log (M r ) Elution volume depends: Native, globular proteins Partially folded molecules Oligomeric state (monomer, dimer, trimer soluble aggregate) Proteins inside detergent micelle: MW of protein + MW of micelle

10 SEC gives different and complementary information to PAGE-SDS Size separation in native or un-native conditions HLTVirE2Superose12prepEndwash001:1_UV1_280nm HLTVirE2Superose12prepEndwash001:1_UV2_260nm HLTVirE2Superose12prepEndwash001:1_UV3_220nm HLTVirE2Superose12prepEndwash001:1_Fractions HLTVirE2Superose12prepEndwash001:1_Inject HLTVirE2Superose12prepEndwash001:1_Logbook mau ml HLT-VirE2 Purification from 200ml cell culture. IMAC purification and Preparative Gel Filtration Superose 12 60x 1.6cm = 200ml Michal Maess from Assaf Friedler lab.

11 How to choose GF type Selectivity fractionation range K av 1 Linear polysaccharides Globular proteins Molecules with different shapes have different selectivity curves Protein 1: 30kDa log (M r ) Protein 2: 80kDa RESIN FR Glob Prot FR Dextrans Sephacryl S kda ND Sephacryl S kda 1-80 kda Vo Vt Sephacryl S kda Sephacryl S kda kda kda

12 Gel Filtration chromatography (GF) Principles of GF Fractionation range Parameters for resolution optimization Use of GF: Purification buffer exchange QC - MW/oligomeric state Protein/protein interaction Examples - Troubleshooting SEC-MALS 12

13 Resolution depends on efficiency and selectivity Low selectivity high efficiency High efficiency can compensate for low selectivity. low efficiency High selectivity high efficiency low efficiency If selectivity is high, low efficiency can be tolerated (if large peak volume is acceptable). 13

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15 Efficiency depends on Efficiency Particle size of matrix (particle size distribution) Packing quality of the column Sample: volume, purity, concentration and viscosity Flow rate (important only for big particle size) Tubing diameter, tubing length and flow path volume Never used longer tubing than necessary 15

16 Peak width depends on particle size Superdex Peptide µm Superdex 30 prep grade µm Retention time (min) Retention time (min) 16

17 Resolution depends on column length Increasing column length increases resolution Retention time (min) Retention time (min) 1 x Superdex Peptide HR 10/ x Superdex Peptide HR 10/30 mau mau SUMO- Atox1 SUMO Atox SUMO- Atox1 SUMO Atox ml Superdex Peptide 60 x 1.6cm ~ 120ml ml Superdex Peptide 100 x 1.6cm ~ 200ml 17 Michal Shoshan from Edith Tshuva lab.

18 Resolution depends on sample volume Column: Superdex Peptide HR 10/ µl Retention volume (ml) Retention volume (ml) 200 µl Retention volume (ml) 400 µl 18

19 Increasing resolution Choose appropriate fractionation range Increase column volume (Connect columns in tandem) Reduce the flow rate Change to a gel with smaller beads (higher efficiency) Volume ~0.5-4% times the expected sample volume Sample volume can be increase if resolution is OK Check the column efficiency Clean and/or re-pack Reduce the sample volume / protein quantity 19

20 SEC Applications Group separations: Desalting, Buffer exchange, Removing reagents (replace dialysis) Purification of proteins and peptides: complex samples, monomer/dimer QC: Size estimation. Size homogeneity: oligomeric state. Impurities. Stability 20 Protein-Protein Interaction

21 Desalting proteins Desalting in a simple column HSA NaCl Column: Sample: Buffer: PD-10 HSA, 25 mg NaCl 0.5M 21 volume Volume for desalting: up to 25% column volume

22 Desalting / Buffer Exchange / Group separation Adjusting ph, buffer type, salt concentration during sample preparation, e.g. before an assay. Removing interfering small molecules: EDTA, Gu.HCl, etc Removing small reagent molecules, e.g. fluorescent labels, radioactive markers Gravity Desalting Columns Multi Spin Desalting Columns Alternative to dialysis or to diafiltration (ultrafiltration at constant retentate) 22 Spin Desalting Columns FPLC Desalting Columns

23 Use buffer that avoid protein precipitation Gali Prag mau HiTrapDesalt10ml001:1_UV1_280nm HiTrapDesalt10ml001:1_Cond HiTrapDesalt10ml001:1_Fractions HiTrapDesalt10ml001:1_Inject HiTrapDesalt10ml001:1_Logbook OD 280nm Conductivity Desalting in the presence of buffer + 100mM NaCl ml HiTrapDesalt10ml002:1_UV1_280nm HiTrapDesalt10ml002:1_Cond HiTrapDesalt10ml002:1_Fractions HiTrapDesalt10ml002:1_Inject HiTrapDesalt10ml002:1_Logbook mau 1000 Desalting in the presence of buffer + 250mM NaCl Waste Waste ml

24 Fractionation of multiple components Separate multiple components in a sample on the basis of differences on their size Best results with samples that contains few components or partially purified samples (polishing step) : Not recommended for proteins with close MW Limited sample volume (0.5-4% of total column volume). Not so suitable if the sample volume is large. Volume can be increase if resolution is still OK (scale up) Flow-rate limitation : Time consuming Removes higher oligomeric states and other aggregates Protein elutes with equilibration buffer (important for storage or buffer exchange) 24

25 Separating dimer and oligomers from monomer mau TIMPSuperdex75Prep120ml001:10_UV1_280nm TIMPSuperdex75Prep120ml001:10_UV2_260nm TIMPSuperdex75Prep120ml001:10_UV3_220nm TIMPSuperdex75Prep120ml001:10_Cond TIMPSuperdex75Prep120ml001:10_Fractions TIMPSuperdex75Prep120ml001:10_Inject 200 Jason Shiran Yulia Shifman lab F ml Secreted Yeast TIMP9 after IMAC Superdex 75 60x1.6cm ~120ml

26 Column size- Sample preparation Desalting and other group separations Column volume: four times the expected sample volume Column length is not so important Preparative separation Column volume: 0.5-4% times the expected sample volume Sample volume can be increase if resolution is OK Column length: cm or more (depends of the resolution: higher length higher resolution) How to reduce volume or concentrate your sample Ultrafiltration Lyophilization Ammonium Sulfate Precipitation (or similar) Reverse elution (bind to absorption column like IEX, HIC, IMAC; and reverse elution) Dialysis vs hygroscopic environment (glycerol, PEG, Sephadex etc) Others 26

27 Quality control: QC size estimation / oligomeric state / impurities / stability Monitor protein prep quality In analytical SEC, the sample volume should be approximately 0.3% of the bed volume to achieve optimal results. Complementary information to PAGE-SDS Gives an estimate of molecular size in native solution Un-native solution: Guanidine HCl, urea, detergents, etc. Precision is not so good as PAGE-SDS For exact MW use SEC-MALS Oligomeric state of the protein / homogeneity / complex. Aggregation profile. Detect presence of impurities. Identify protein interaction partners and interaction conditions 27

28 QC: monitoring size-homogeneity changes during storage or stress conditions Aggregation Evaluate tendency to aggregate and quantity aggregates Degradation Evaluate tendency to degradetion and quantity of degraded forms

29 A new trend: UHPLC (Ultra high-performance liquid chromatography) The trend is towards smaller particles of < 2 μm, with the use of ultra high-performance liquid chromatography (UHPLC) systems for even faster separations in high-throughput mode. But: Very high back pressure Demands specific equipment Loss of resolution due to dead volumes Heat generation and shear stress at high flow rates could affect proteins

30 Gel Filtration chromatography (GF) Principles of GF Fractionation range Parameters for resolution optimization Use of GF Troubleshooting Examples SEC-MALS 30

31 Troubleshooting Lower yield than expected Protease degradation of the protein Adsorption to filter, valves or top of the column Non-specific adsorption Sample precipitate MW of protein is not as expected Oligomerization state of the protein is different Protein bounds to another protein or complex Unfolded or naturally unfolded protein Protein has changed during storage Ionic or Hydrophobic interactions with the matrix Protein precipitate Very broad peak elution Different oligomeric states or protein aggregation Sticky protein Non specific adsorption to matrix Protein is part of complex with different sizes Overloading Peak of interest is poorly resolved Sample volume is too high Column length is not enough Poor selectivity or efficiency of the column Flow rate too high Column is dirty or not well packed Viscous sample 31

32 Case study: HLT-p53CT Capture: IMAC Affinity start with pellet of 1.5L culture Ni-Sepharose FF 14ml Ronen Gabizon from Assaf Friedler lab. mau HLTp53CTNiNTA16ml004:1_UV1_280nm HLTp53CTNiNTA16ml004:1_UV2_260nm HLTp53CTNiNTA16ml004:1_Conc HLTp53CTNiNTA16ml004:1_Fractions HLTp53CTNiNTA16ml004:1_Inject HLTp53CTNiNTA16ml004:1_Logbook 2500 Load + 10cv 0%B + 3cv 8%B + 4cv 15%B + 4cv 100%B POOL 17-22: 3.5OD x 35ml ~ 276mg /15/2018 F4 Waste ml

33 mau 150 Case study: HLT-p53CT Intermediate Cation Exchange HLTp53CTHiTrapSP5mlml005:1_UV1_280nm HLTp53CTHiTrapSP5mlml005:1_UV2_260nm HLTp53CTHiTrapSP5mlml005:1_Cond HLTp53CTHiTrapSP5mlml005:1_Conc HLTp53CTHiTrapSP5mlml005:1_Fractions HLTp53CTHiTrapSP5mlml005:1_Inject HLTp53CTHiTrapSP5mlml005:1_Logbook After TEV protease cleavage ON 4ºC SP-Sepharose FF 5ml /15/2018 F ml

34 Case study: HLT-p53CT Polishing SEC column Column: Sephacryl S100 prep. 960 x 26mm (~500ml) 6ml/fract. Ni column TEV protease cleavage ON dilution CEIX concentration & GF Ronen Gabizon from Assaf Friedler lab. HLTp53CTSephacrylS100of500ml004:1_UV1_280nm HLTp53CTSephacrylS100of500ml004:1_UV2_260nm HLTp53CTSephacrylS100of500ml004:1_Fractions HLTp53CTSephacrylS100of500ml004:1_Inject HLTp53CTSephacrylS100of500ml004:1_Logbook mau 150 POOL Fractions around 23 and 32 are higher MW impurities F ml

35 120617M1605Superose12Anal001:1_UV1_280nm M1605Superose12Anal001:1_UV2_260nm M1605Superose12Anal001:1_Fractions M1605Superose12Anal001:1_Inject M1605Superose12Anal001:1_Logbook Increasing resolution Example: Pegylated protein Superose12Prep3columns500ml001:1_UV1_280nm Superose12Prep3columns500ml001:1_UV2_260nm Superose12Prep3columns500ml001:1_Fractions Superose12Prep3columns500ml001:1_Inject Superose12Prep3columns500ml001:1_Logbook mau 150 mau 150 Vo F Waste ml Superose 12 analytical 30 x 1cm = 23ml column Load : ~1mg protein ml Superose 12 preparative 3 tandem columns 250 x 1.6cm = 502ml column Load : ~25mg protein / 5ml How can we get better separation between 2 and 3?? Can we scale-up protein loading to separate 1 from 2 and 3??

36 CMV-Viral Glycoprotein Extracellular Expression in Insect Cells Without DTT / boiling Pool fractions 8-17 Before 6 7 M kDa Ultrafiltration and SEC purification in tandem Superose 12 & Superdex x 1.6cm each ~ 400ml total mau 100 Protein MW (kda) Ve (ml) Darpin:10_UV1_280nm Darpin:10_UV2_260nm Darpin:10_Fractions Darpin:10_Inject Darpin:10_Logbook Thyroglob SEC-MALS: Superdex 200 analytical column Peak1: Total mass = 850±30 kda Protein mass = 440±20 kda Ration = ~1:1 Peak2: Total mass = 85±5 kda Protein mass = 2.6±0.2 kda Results Fitting Protein Molar Mass 1 Protein Molar Mass 2 LS UV RI 80 Ferritin x10 60 Catalase x BSA Chymotr Molar Mass (g/mol) 4 1.0x cyncy town 1 36 Peak Merlin 1 Peak Waste Waste Waste Waste ml Cincy Town volume (ml)

37 Complex formation Example: Leptin and Leptin Receptor Superdex75prep002:1_UV3_220nm Superdex75prep002:1_Fractions Superdex75prep002:1_Inject Superdex75prep002:1_UV3_220nm1 Superdex75prep002:1_UV3_220nm2 Superdex75prep002:1_Logbook mau Superdex x1.6cm column - Buffer: 20mMTrisHCl ph8.0 50mMNaCl 0.02%NaN Complex: Leptin + Receptor Leptin alone Receptor alone Waste ml

38 Case study: Native Unfolding Protein HTL - A natively unfolded proline-rich domain in ASPP2 that regulates its protein interactions by intramolecular binding to the Ank-SH3 domains. Shahar Rotem et al. JBC Friedler lab mau kda FoldIndex : a simple tool to predict whether a given protein sequence is intrinsically unfolded. Jaime Prilusky, Clifford E. Felder, Tzviya Zeev-Ben-Mordehai, Edwin Rydberg, Orna Man, Jacques S. Beckmann, Israel Silman, and Joel L. Sussman, 2005, Bioinformatics. HTL435aaSuperdex200prep500mlB005:11_UV3_220nm HTL435aaSuperdex200prep500mlB005:11_Logbook 435 aa MW: 49.7kD Superdex 200 prep. 100x2.6cm : trimer?? Analytical ultracentrifugation: monomer 50 HTL 435aa ml

39 Size Exclusion Chromatography - Multi Angle Light Scattering SEC-MALS Size exclusion chromatograph in line with multi angle light scattering, added value to characterize proteins mass and shape in native solution conditions Calculating Mw and radius from the light scattering equations much more accurate. Calculate the Mw during the elution peaks- detect homogeneity sample. Detect low amount of aggregation large molecules amplify the intensity of LS. Useful for protein/protein or protein/ligand interaction

40 Quasi Elastic Light Scattering (QELS) / Dynamic Light Scattering (DLS) Measures the time-dependent fluctuations in the intensity of the scattered light caused by random motion of the macromolecules in the solution. The fluctuations are related to the rate of diffusion which is related to the radius of the molecule. Stokes-Einstein equation: R- radius k- Boltzmann constant T-temperature D- diffusion coefficient η- viscosity

41 Radius of gyration vs. hydrodynamic radius Dynamic Light Scattering: R h or Hydrodynamic Radius radius of a sphere with the same diffusion coefficient as our sample. lower limit ~ 0.5 nm R h Static Light Scattering: RMS Radius or R g mass averaged distance of each point in a molecule from the molecule s center of gravity. lower limit 10 nm

42 Calculating mass of intrinsically disordered proteins SEC alone Normalized absorbance at 280 nm An intrinsically disordered protein (17 kda) ml (~45 kda) Volume (ml) Predicted elution volume according to calibration curve

43 Calculating mass of intrinsically disordered proteins 1.0 LS UV An intrinsically disordered protein (17 kda) Ve: kDa according to calibration curve 10 6 Normalized intensity kDa Predicted elution volume according to calibration curve Molar mass (Da) Volume (ml)

44 LS is very sensitive for aggregates Aggregate (0.3%) Monomer (99.7%) 1.0 LS UV Normalized intensity kda Molar mass (Da) Volume (ml)

45 Mass (Da) Protein aggregation induced by a specific molecule 1.0 Alone + M (low conc.) + M (high conc.) 10 8 Normalized UV intensity Elution volume (ml)

46 Mass (kda) P53 CTD oligomerization WT- tetramer, L344A mutation inhibits P53 CTD oligomerization P53 CTD (11 kda) WT & L344A mutant Normalized UV intensity WT L344A Elution volume (ml)

47 Molar mass Hydrodynamic radius Studying protein modifications Use to study protein modification such as glycosylation and pegylation. Can be used to characterize number of modifications. Can be used to study structural changes in modified proteins. 47 Volume Volume

48 Molar masses for two distinct ADC (antibody-drug conjugates) formulations are determined using SEC-MALS analysis WYATT Technology 48

49 Studding polymers using MALS Mass (Da) 1.0 Polymer sample LS RI Mass Normalized intensity Elution volume (ml)

50 Downstream application in industry Preparative column: measure aggregation percent UV Absorbance Elution time (min) Analytical SEC-MALS : 1.0 5% 0.5% 0.01% Fast SEC-MALS experiment Normalized intensity Volume (ml)

51 Static light scattering to characterize membrane proteins in detergent solution D.J. Slotboom et al. / Methods 46 (2008) accuracy of the determined molecular masses. For the determination of the absolute molecular mass of membrane proteins in protein/detergent/lipid micelles The size exclusion column is used to physically separate aggregates/empty micelles from the protein of interest, and to ensure that the protein is dissolved in the correct buffer. The elution volume is not included in the calculations. The technique provides very similar information as sedimentation equilibrium centrifugation, with similar

52 Summary of SEC-MALS SEC-MALS is a useful tool to determine protein shape and mass, characterize oligomerization/aggregation and verify protein purity. Very sensitive to presence of aggregates Additional information: modifications (glycosilation, pegylation, etc) Choose a good column for best separation of the sample for achieving accurate results. Limitation: can detect low amount of large macromolecules but needs high concentration of small macromolecules Requires longer equilibration time

53 Field flow fractionation (FFF) Some molecules are highly hydrophobic, making them incompatible with fractionation via size-exclusion chromatography (SEC). Field flow fractionation (FFF) separates macromolecules and nanoparticles by size without a stationary phase, eliminating most of the non-ideal surface interactions prevalent in SEC. In an Asymmetric-Flow FFF separation channel, macromolecules and nanoparticles are gently pushed against a semipermeable membrane by crossflow. Smaller particles diffuse back up towards the center of the channel. Laminar channel flow induces a parabolic flow velocity profile, causing smaller particles to elute earlier. 53

54 Why choose gel filtration? Advantage Separates by size Complementary to IEX and HIC Very gentle, high yields Works in any buffer solution Removes aggregates Fast for buffer exchange Mostly use in a final polishing step Disadvantage Limited sample volume Poor resolution in a complex mixture Flow-rate limitation time consuming Sample is diluted during elution Poor selectivity compared with SDS-PAGE Not efficient in capture or intermediate steps Mandatory for QC Complementary results than PAGE-SDS 54

55 Dual functionality: size exclusion, and binding chromatography - Capto Core GE For intermediate purification and polishing of viruses and other large biomolecules (M r > ) in flow-through mode. Efficient capture of contaminants (HCP, DNA) Target molecules are collected in the FT Significantly improved productivity and higher flow rates compared with GF Octylamine ligands inside the core of beads: both hydrophobic and positively charged, resulting in a highly efficient multimodal binding of various contaminants small enough to enter the core. The multimodal ligand ensure strong binding with most impurities over a wide range of ph and salt concentrations

56 Virus HCP Dual functionality: size exclusion, and binding chromatography Capto Core 700 Sample: Influenza H1N1 cultivated in MDCK cells, concentrated, and diafiltrated on an Mr hollow-fiber filter to 20 mm Tris, 150 mm NaCl, ph 7.5 Columns: Tricorn 10/600 packed with Sepharose 4 Fast Flow, CV 47 ml Sample loads: Sepharose 4 Fast Flow, 0.1 CV (4.7 ml) Flow velocities: Sepharose 4 Fast Flow, 30 cm/h Sepharose 4 Fast Flow Virus HCP Columns: Tricorn 5/50 packed with Capto Core 700, CV 1 ml Sample loads: Capto Core 700, 10 CV (10 ml) Flow velocities: Capto Core 700, 100 cm/h Buffer: 20 mm Tris, 150 mm NaCl, ph 7.5 Cleaning-in-place (CIP)/elution: Capto Core 700, 30% isopropanol in 1 M NaOH Capto Core 700 The sample load for Sepharose 4 Fast Flow was 0.1 CV The equivalent load for the Capto Core 700 was 10 CV

57 Process Development Platform AAV purification process

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61 Adeno Associated Viruses (AAVs) purification Separation of empty from full capsids by AEIX

62 Western blot: AAV9 on Hi Trap Q 1ml - lysate sample mau Virus with DNA ( full ) Capsid without DNA ( empty ) Buffers: Wash: 20mM Tris ph9 5% sorbitol Elution: 20mM Tris ph9, 5% sorbitol, 500mM NH 4 Ac Final wash: 1M NaCl AAV9 lysate HiTrap Q 1ml001:10_UV1_280nm AAV9 lysate HiTrap Q 1ml001:10_UV2_260nm AAV9 lysate HiTrap Q 1ml001:10_Cond AAV9 lysate HiTrap Q 1ml001:10_Conc AAV9 lysate HiTrap Q 1ml001:10_Fractions 100% 280 nm 260 nm % Elution buffer Conductivity Capsid proteins qpcr for viral DNA Fraction GC/mL E E E E E % Waste Waste

63 mau AAV9 lysate HiTrap Q 1ml001:10_UV1_280nm AAV9 lysate HiTrap Q 1ml001:10_UV2_260nm AAV9 lysate HiTrap Q 1ml001:10_Cond AAV9 lysate HiTrap Q 1ml001:10_Conc AAV9 lysate HiTrap Q 1ml001:10_Fractions AAV9 lysate on AIEX Buffers: A- 20mM TRIS ph9 5% sorbitol B- 20mM TRIS ph9 5% sorbitol 500mM N 100%B (500mM NH 4 Ac) %B (~250mM NH 4 Ac) Waste Waste Waste ml

64 AAV9 Lysate on AIEX mau AAV9 lysate HiTrap Q 1ml 001:10_UV1_280nm AAV9 lysate HiTrap Q 1ml 001:10_UV2_260nm AAV9 lysate HiTrap Q 1ml 001:10_Cond AAV9 lysate HiTrap Q 1ml 001:10_Conc AAV9 lysate HiTrap Q 1ml 001:10_Fractions Buffers: A- 20mM TRIS ph9 5% sorbitol B- 20mM TRIS ph9 5% sorbitol 500mM NH4Ac 100%B (500mM NH 4 Ac) 1500 mau AAV9 lysate HiTrap Q 1ml 001:10_UV1_280nm AAV9 lysate HiTrap Q 1ml 001:10_UV2_260nm AAV9 lysate HiTrap Q 1ml 001:10_Cond AAV9 lysate HiTrap Q 1ml 001:10_Conc AAV9 lysate HiTrap Q 1ml 001:10_Fractions %B (~250mM NH 4 Ac) 0 Waste 4 Waste Waste ml 500 Fractions 7-11 were loaded on capto core700 5%B (~25mM NH 4 Ac) 0 2 Waste 3 Waste 4 Waste Waste ml