CHROMATOGRAPHY. Duarte Miguel Prazeres
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1 CHROMATOGRAPHY Duarte Miguel Prazeres Instituto Superior Técnico, 2009
2 CHROMATOGRAPHIC MATRICES Material Organic Polysaccharides Other polymers Example (trade names) Dextran (Sephadex, Sephacryl ) Agarose (Sepharose, Biogel ) Cellulose (Cellufine, Sephacel, Avicell ) Polyacrylamide Polyvinil (Fractogel, Toyopearl ) Polystyrene/divinilbenzene (Source, Poros, Supelcogel, Dowex, PolyBio ) Polystyrene (Combigel ) Trisacryl (Trisacryl ) Inorganic Silica (Spherosil, Supelcosil, Kromasil, Zorbax, Lichroprep, Lichrospher ) Diatomaceous earth (Celite, Kieselghur) Calcium phosphate Porous glass Carbon (Pulsorb, Ambersorb, Flitrasorb ) Alumino silicate (zeolites, Fuller s earth) Magnesium silicate (Florisil ) Composite Polyacrylamide -agarose (Ultrogel ) Dextran-bisacrylamide Polystyrene/polyacrylamide (HyperD ) Porous silica-dextran (Zephyr, Spherodex ) Quartz/agarose/dextran (Streamline )
3 STATIONARY PHASES
4 STATIONARY PHASES
5 STATIONARY PHASES
6 CHROMATOGRAPHIC SYSTEM t 9 Legend 1 feed 2 injector 3 eluent tanks 4 pumps 5 mixer 6 chromatographic bed 7 detectors 8 recorder and control system 9 fraction colector 10 chromatogram
7 ANALYTIC vs PREPARATIVE CHROMATOGRAPHY Analytic chromatography Preparative chromatography Relative absorbance 280 nm target impurity Ammonium sulphate (M) Time (min) -0.5
8 ANALYTICAL CHROMATOGRAPHY
9 ANALYTICAL CHROMATOGRAPHY
10 PREPARATIVE CHROMATOGRAPHY
11 PREPARATIVE CHROMATOGRAPHY
12 PREPARATIVE CHROMATOGRAPHY
13 TRUCK RACING A rate controlled separation u A > u B L L = L min L < L min
14 RATE CONTOLLED SEPARATIONS u A > u B L A + B column leito fixo L porosidade intra-particular, ε p porosidade inter-particular, ε e
15 TRUCK RACING A rate controlled separation poor separation L separation L excellent separation L
16 SIZE EXCLUSION CHROMATOGRAPHY K D = 0 K D = 1 Distribution coefficient: C K D = C Concentration inside the pores Concentration outside the particle The specific selection curve of Cellufine GH-25 K D 1: Glycine 75 2: (Gly) : (Gly) : (Gly) : Calcium pantothenate 477 6: Vitamin B : insulin B chain 3495
17 SIZE EXCLUSION CHROMATOGRAPHY Typical elution pattern Column volumes (CV) Desalting A280 nm (ms/cm) time (h) Protein purification Handbook, Amersham Pharmacia biotech
18 SIZE EXCLUSION CHROMATOGRAPHY Polishing of zz-igf A280 nm aggregates dimers Polishing of monoclonal IgG1 Protein purification Handbook, Amersham Pharmacia biotech
19 ION EXCHANGE CHROMATOGRAPHY matrix ligand Cation exchange SP- sulfopropyl (strong) CM- carboxymethyl (weak) (CH 2 ) 3 OSO 3 - X + CH 2 COO - X + Anion exchange DEAE- diethyl amino ethyl (weak) QAE- quaternary amino ethyl (strong) (CH 2 CH 2 )N + H(CH 2 CH 3 ) 2 Y - (CH 2 CH 2 )N + (CH 2 CH 3 ) 3 Y - X +, Y - - counter-ions Anion exchange Cation exchange Capacity weak strong Capacity strong weak ph ph
20 ION EXCHANGE CHROMATOGRAPHY Gradient elution Step elution Protein purification Handbook, Amersham Pharmacia biotech
21 ION EXCHANGE CHROMATOGRAPHY ph effect Cation exchange Surface net charge Anion exchange Protein purification Handbook, Amersham Pharmacia biotech
22 ION EXCHANGE CHROMATOGRAPHY Anion exchange capture of recombinant α-amylase from an E. coli extract A280 nm α-amylase Conductivity 1 M NaCl 0 M NaCl Fraction Feed Flow through 1st Peak (α-amylase) 2nd Peak ng DNA/µl Protein purification Handbook, Amersham Pharmacia biotech
23 ION EXCHANGE CHROMATOGRAPHY Anion exchange capture of de-acetoxycephalosporin C synthase (pi = 4.8)
24 HYDROPHOBIC INTERACTION CHROMATOGRAPHY Gradient Elution Step Elution Protein purification Handbook, Amersham Pharmacia biotech
25 HYDROPHOBIC INTERACTION CHROMATOGRAPHY Purification of a recombinant antigen by HIC 1 M (NH 4 ) 2 SO 4 antigen 0.5 M (NH 4 ) 2 SO 4 impurities 0 M (NH 4 ) 2 SO 4 Protein purification Handbook, Amersham Pharmacia biotech
26 HYDROPHOBIC INTERACTION CHROMATOGRAPHY Purification of de-acetoxycephalosporin C synthase by HIC DAOCS 1.6 M (NH 4 ) 2 SO 4 0 M (NH 4 ) 2 SO 4 Protein purification Handbook, Amersham Pharmacia biotech
27 HYDROPHOBIC INTERACTION CHROMATOGRAPHY Gradient elution
28 AFFINITY CHROMATOGRAPHY affinity interactions molecular recognition between two complementary biological entities ligand matrix target molecule(s) spacer arm binding wash elution (ph, ionic strength, competitor)
29 AFFINITY CHROMATOGRAPHY 1. Column equilibration 2. Feed injection (binding of target molecule) 3. Washing of unbound material 4. Elution target molecule Protein purification Handbook, Amersham Pharmacia biotech
30 AFFINITY CHROMATOGRAPHY Ligand Lectins (e.g. Concanavalin A) Complementary solute Glycoproteins Heparin Cibacron blue Iminodiacetic acid + Metals Coagulation factors (VII, IX, XI, XII) Growth hormones Lipopolyproteins Albumin Hormones and growth factors Interferon Enzymes w/ NAD, NADPH co-factors Proteins w/ histidines and cysteines Protein A, Protein G DNA Biotin Lysine Immunoglobulin G Monoclonal antibodies Proteins (e.g Zinc fingers) Avidin, Streptavidin Plasminogen Glycoproteins DNA, RNA
31 CONCANAVALIN A -SUGAR Concanavalin A binds to molecules with α-d-mannose, α-d-glucose Elution is made using a buffer w/ a competitor (e.g. mannose) Concanavalin A glycoprotein mannose matrix sugar
32 HEPARIN-PROTEIN PROTEIN Heparin sulfated glycosaminoglycan, natural anticoagulant Binds strongly to different proteins (AT III, coagulation factors, etc.) 2 M NaCl 0 M NaCl
33 CIBACRON BLUE - PROTEIN Cibacron a dye with affinity towards enzymes w/ co-factors and proteins such as albumin and interferon binding to the matrix A280 albumin 1.5 M KCl
34 IMAC immobilized metal affinity chromatography Imino-diacetic acid forms complexes with metalic ions (Zn 2+, Ni 2+ ) Ligands adequate for the purification of proteins which form metalic complexes via specific amino acids (histidine and cysteine) Ni histidines Elution is made with imidazol
35 IMAC Histidine tag A280 Ni 9 mm imidazol interferon
36 PROTEIN G-IgG Fc region IgG ph7 ph4
37 ZINC FINGERS The coordination of 2 cysteines and 2 histidines with a Zn atom keeps the finger structure together α helix β sheet Cysteines Zinc Histidines
38 TRANSCRIPTION FACTOR IIIA Protein with 9 zinc fingers The protein does not function without Zinc The α helix interacts through the major groove Dedo 1 Dedo 2 Dedo 3
39 BIOTIN -STREPTAVIDN Biotin (also known as vitamin H or B7) Streptavin a 60,00 Dalton tetrameric protein biotin
40 CASE STUDY:PLASMA FRACTIONATION PLASMA Albumin IgG Factor VIII Factor IX
41 CASE STUDY:PLASMA FRACTIONATION Albumin IgG Factor VIII Factor IX
42 PLASMA FRACTIONATION Plasma Size exclusion (desalting) Anion exchange (weak) Cation exchange (strong) Anion exchange (strong) Cation exchange (strong) Size exclusion Cation exchange (strong)
43 SIZE EXCLUSION proteins salts Objective: remove salts 1. Solid phase: Sephadex G-25C 2. Buffer: sodium acetate ph 7, 5 mm 3. Column: 60 x 60 cm 4. Feed volume: 25 l x 15 cycles = 375 l 5. v s = 300 cm/h 6. Total volume of buffer: 200 l x 15 = 3000 l 7. Total process time: 4.2 hours
44 ANION EXCHANGE Objective: separate Albumin from IgG and other proteins albumin IgG plasma 1. Solid phase: DEAE Sepharose FF 2. Buffer 1: sodium acetate ph 5.2, 20 mm 3. Buffer 2: sodium acetate ph 4.5, 25 mm 4. Buffer 3: sodium acetate ph 4.0, 150 mm 5. Column: 100 (id) x 15 cm 6. Feed volume: 275 l x 3 cycles = 825 l 7. v s = 120 cm/h 8. Total buffer volume: 1100 l x 3 = 3300 l 9. Total process time: 5.0 hours
45 CATION EXCHANGE albumin other proteins Objective: separate Albumin from other proteins plasma 1. Solid phase: CM Sepharose FF 2. Buffer 1: sodium acetate ph 4.5, 25 mm 3. Buffer 2: sodium acetate ph 5.5, 110 mm 4. Buffer 3: sodium acetate ph 8.0, 400 mm 5. Column: 100 (id) x 15 cm 6. Feed volume: 165 l x 3 cycles = 495 l 7. v s = 120 cm/h 8. Total buffer volume: 1300 l x 3 = 4000 l 9. Total process time: 5.1 hours
46 SIZE EXCLUSION albumin aggregates plasma Objective: remove Albumin aggregates 1. Solid phase: Sephacryl S-200 HR 2. Buffer: sodium acetate ph 6.0, 20 mm 3. Column: 80 (id) x 30 cm 4. Feed volume : 17,3 l x 9 cycles = 156 l 5. v s = 40 cm/h 6. Total buffer volume: 140 l x 9 = 1260 l 7. Total process time: 32.6 hours
47 PROCESS VOLUMES Plasma (375 l) Size exclusion 825 l Anion exchange 495 l Cation exchange 156 l Size exclusion 488 l
48 PROCESS DURATION Size exclusion 4.2 h Anion exchange 5 h Cation exchange 5.1 h Size exclusion 32.6 h 47 h
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