Bioseparation. From Microanalysis To Plant-Scale Purification PB-0065E

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Cynthia Carr
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1 From Microanalysis To Plant-Scale Purification Bioseparation Selection Guide Comparison of Separation Mode s/packing Materials Ion Exchange s/media Size Exclusion s Reversed-phase s/packing Materials Preparative Systems Biochromatography Devices/s Glass s PB-6E

2 i Bioseparation products catalog Selection Guide

3 Comparison of Separation Mode IgG Albu Albu Transferrin Transferrin IgG N8E Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing. M NaCl -%B (- ), -%B (- ) :. ml/ Temperature : C : UV at 8 nm Injection : L ( L/mL) N866A 6 Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl :. ml/ Temperature : ambient ( C) : UV at 8 nm Injection : L ( L/mL) P8A NaN Mouse monoclonal IgG (Anti-human IgG) P8A Mouse monoclonal IgG (Anti-human IgG) NaN Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl -%B (-8 ) :. ml/ Temperature : C : UV at nm Injection : L (. mg/ml) Eluent :. M KHPO-KHPO (ph 7.) :.7 ml/ Temperature : ambient ( C) : UV at nm Injection : L (. mg/ml). Rabbit IgG. Mouse IgG Fc fragment Mouse IgG Fc fragment R86D Eluent :. M KHPO-KHPO (ph 6.9) containing. M NaCl :. ml/ Temperature : ambient (7 C) : UV at nm Injection : L (. mg/ml) R869B Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -%B (- ) :. ml/ Temperature : 7 C : UV at nm Injection : L (. mg/ml) Bioseparation products catalog

4 6 N8B 6 N86L 6 * * MW Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing. M NaCl -%B (- ), -6%B (-6 ) :. ml/ Temperature : C : UV at nm Injection : L. Myoglobin 7,). Insulin (Bovine),7). Neurotensin,67). Tetraglycine 6). Glycine 7) Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl/acetonitrile (7/) :.7 ml/ Temperature : ambient ( C) Injection : UV at nm : L N88B 6 Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -%B (- ), -%B (- ), %B (-6 ) :. ml/ Temperature : 7 C : UV at nm Injection : L * undigested BSA * G967A G96A Eluent : methanol/ mm NHHPO (/9) :. ml/ Temperature : 7 C : FLS at Ex. nm, Em. nm Injection : L (. pmol/ml) Sample : PA-Sugar Chain Series, manufactured by TAKARA BIO INC. Eluent : methanol/ mm NHHPO (8/) :. ml/ Temperature : 7 C : FLS at Ex. nm, Em. nm Injection : L (. pmol/ml) Sample : PA-Sugar Chain Series, manufactured by TAKARA BIO INC. Bioseparation products catalog

5 N8G Eluent : A) mm Tris-HCl (ph 8.) containing.7 M NaCl B) mm Tris-HCl (ph 8.) containing. M NaCl -%B (- ) :. ml/ Temperature : C : UV at 6 nm Injection : L Plasmid pbr Hae digest (8-87 bp) Plasmid pbr (,6 bp) N86F Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl 7-8%B (- ), 8%B (- ) :. ml/ Temperature : C : UV at 6 nm Injection : L Plasmid pbr (,6 bp) Plasmid pbr Hae digest (8-87 bp) P867A Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl :.7 ml/ Temperature : ambient ( C) : UV at 6 nm Injection : L UV MS nt nt Int.( x ) -pugg AGU GUG ACA AUG GUG UUG- ( nt, MW 689.) -pugg AGU GUG ACA AUG GUG UUG U- ( nt, MW 796.) MIC (m/z 7., m/z 798.) nt nt FA Courtesy of M.Yamada, SHIMADZU CORPORATION Eluent : A) mm DBAA* (ph 7.) B) mm DBAA* (ph 7.)/acetonitrile (/) 6-7%B (- ) :. ml/ Temperature : : UV at 6 nm and ESI-negative mode Injection : L ( nmol/ml) Instrument : LC) Shimadzu Proence MS) Shimadzu LCMS * di-n-butyla-acetic acid Bioseparation products catalog

6 s/packing Materials BioPro Bioseparation products catalog

Trypsin inhibitor N86B N87C N87B N86E N88C N88B N86A N86D 7 7 7 7 7..... Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl -8%B (- ) :.

7 YMC-BioPro QA / YMC-BioPro SP AU.. BioPro QA % breakthrough : YMC-BioPro QA X.6 mmi.d. Brand T (porous Q type) X.6 mmi.d. Brand G (porous Q type) X. mmi.d. Linear velocity : 8 cm/hr Equilibration buffer : mm Tris-HCl (ph 8.6) Elution buffer : mm Tris-HCl (ph 8.6) containing. M NaCl : UV at 8 nm Sample : mg/ml Bovine serum albu (BSA) in equilibration buffer. Ovalbu. Trypsin inhibitor N86B N87C N87B N86E N88C N88B N86A N86D Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl -8%B (- ) :. ml/ (8 cm/hr for.6 mmi.d., cm/hr for. mmi.d.) Temperature : C : UV at 8 nm Injection : L Bioseparation products catalog 6

YMC-BioPro QA-F / YMC-BioPro SP-F AU... ml/ ( cm/hr).8-. MPa. N7K AU.. ml/ (8 cm/hr).-. MPa N76E 6. Ribonuclease A. Cytochrome c. Lysozyme Eluent Temperature Injection : A) mm KHPO-KHPO (ph 6.

8 YMC-BioPro QA-F / YMC-BioPro SP-F AU... ml/ ( cm/hr).8-. MPa. N7K AU.. ml/ (8 cm/hr).-. MPa N76E 6. Ribonuclease A. Cytochrome c. Lysozyme Eluent Temperature Injection : A) mm KHPO-KHPO (ph 6.8) B) mm KHPO-KHPO (ph 6.8) containing. M NaCl -%B (- ) for YMC-BioPro SP-F -%B (-6 ) for YMC-BioPro SP : C : UV at nm : L Eluent : A) mm Tris-HCl (ph 8.) containing.7 M NaCl B) mm Tris-HCl (ph 8.) containing. M NaCl -%B (- ) :. ml/ (8 cm/hr) Temperature : C : UV at 6 nm Injection : L (. mg/ml) N8G P8A P8B NaN NaN MAb MAb Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl -%B (-6 ) :. ml/ (6 cm/hr) Temperature : C : UV at nm Injection : L (. mg/ml) Sample : Mouse monoclonal IgG anti-human IgG (Purified by DEAE chromatography, containing NaN) 7 Bioseparation products catalog

9 tr VF 6 8 tr VG 6 8 Eluent Initial gradient conc. Gradient slope Temperature Injection Sample : A) mm MES-NaOH (ph.6) B) mm MES-NaOH (ph.6) containing. M NaCl : %B (7 mm NaCl :.%B/ (. mm NaCl) : 8 cm/hr (. ml/ for X.6 mmi.d.,.78 ml/ for X. mmi.d.) : : UV at 8 nm : L : Humanized monoclonal IgG ( mg/ml) Lot. E Lot. D : YMC-BioPro SP-F m, X.6 mmi.d. Eluent : A) mm NaHPO-NaHPO (ph 6.) B) mm NaHPO-NaHPO (ph 6.) containing. M NaCl -%B (.- ) :. ml/ (8 cm/hr) Temperature : : UV at nm Injection : L Sample : monoclonal antibody (IgG) Lot. C Lot. B Lot. A Bioseparation products catalog 8

10 BioPro SmartSep Q/S DBC (mg/ml-resin) 8 6 : X. mmi.d. : cm/hr (. ml/) Temperature : Insulin :,8) Lysozyme :,) Human Polyclonal IgG :,) DBC (mg/ml-resin) Equilibration buffer Elution buffer Temperature Sample : X. mmi.d. : mm citric acid-naoh (ph.) : Equilibration buffer containing. M NaCl : -8 cm/hr ( ml/) : ambient ( ) : UV at 8 nm :. mg/ml human polyclonal IgG in equilibration buffer 6 8 Linear velocity (cm/hr) 9 Bioseparation products catalog

11 K89P : X. mmi.d. Eluent : A) mm NaHPO-NaHPO (ph 6.8) B) mm NaHPO-NaHPO (ph 6.8) containing. M NaCl -%B (- column volumes) : 6 cm/hr (. ml/) Temperature : : UV at nm Injection : ml ( mg Proteins) Sample :. Ribonuclease A (. mg/ml). Cytochrome c (. mg/ml). Lysozyme (. mg/ml) K86P K86P 6 7 : X. mmi.d. Eluent : A) mm citric acid-naoh (ph.) B) mm citric acid-naoh (ph.) containing. M NaCl -%B, column volumes : 8 cm/hr (.9 ml/) Temperature : ambient : UV at 8 nm Sample : Anti-h TNF alpha IgG (Purified by Affinity chromatography) Injection :. ml (. mg IgG) Bioseparation products catalog

12 BioPro Ion Exchange Media * Dynamic binding capacities were detered at % breakthrough under following conditions: : X.6 mmi.d. : 8 cm/hr (. cm/) for anion-exchange media Equilibration buffer : mm Tris-HCl (ph 8.6) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml BSA in equilibration buffer : UV at 8 nm for cation-exchange media Equilibration buffer : mm Glycine-NaOH (ph 9.) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml Lysozyme in equilibration buffer : UV at nm Equilibration buffer Elution buffer Sample : X. mmi.d. : mm Glycine-NaOH (ph 9.) :. M NaCl in equilibration buffer :. mg/ml Lysozyme in equilibration buffer : UV at nm : X.6 mml.d : 8 cm/hr for anion-exchange media Equilibration buffer : mm Tris-HCI (ph 8.6) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml BSA in equilibration buffer : UV at 8 nm for cation-exchange media Equilibraiton buffer : mm Glycine-NaOH (ph 9.) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml Lysozyme in equilibration buffer : UV at nm Bioseparation products catalog

13 : BioPro S7 X. mmi.d. : 8 cm/hr (.6 ml/) Equilibration buffer : mm Glycine-NaOH (ph 9.) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml Lysozyme in equilibration buffer Temperature : ambient : UV at nm DBC was detered at % breakthrough Eluent Gradient Temperature Injection Sample : BioPro S7 X. mmi.d. : A) mm NaHPO-NaHPO (ph 6.8) B) mm NaHPO-NaHPO (ph 6.8) containing. M NaCl : -%B (-6 ; Linear) : 8 cm/hr (.9 ml/) : : UV at nm : L :. Ribonuclease A,. Cytochrome c,. Lysozyme (. mg/ml) 6 8 : BioPro Q7 X.6 mmi.d. Equilibration buffer : mm Tris-HCl (ph 8.6) Elution buffer :. M NaCl in equilibration buffer : 8 cm/hr (. ml/) Sample :. mg/ml BSA in equilibration buffer Temperature : : UV at 8 nm DBC was detered at % breakthrough Eluent Gradient Temperature Injection Sample : BioPro Q7 X.6 mmi.d. : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl : -8%B (-6 ; Linear) : 8 cm/hr (. ml/) : : UV at nm : L :. Transferrin (. mg/ml),. Trypsin inhibitor (. mg/ml) Bioseparation products catalog

ml/) Temperature : ambient : UV at 8 nm Injection : ml (about mg Protein) 97.kDa 66.kDa.kDa 9.kDa.kDa.kDa mv 7 7.. Eluent :. M KHPO-KHPO (ph 6.

14 mv Eluent :.M KHPO-KHPO (ph 6.9) containing. M NaCl :. ml/ Temperature : ambient : UV at 8 nm A98J 6 Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl %B (- ), %B (- ), 9%B (- ) : 8 cm/hr (. ml/) Temperature : ambient : UV at 8 nm Injection : ml (about mg Protein) 97.kDa 66.kDa.kDa 9.kDa.kDa.kDa mv Eluent :. M KHPO-KHPO (ph 6.9) containing. M NaCl :. ml/ Temperature : ambient : UV at 8 nm.. Eluent :.M KHPO-KHPO (ph 6.9) containing. M NaCl :.7 ml/ Temperature : ambient : UV at 8 nm Injection : ml (about. mg IgY) 97.kDa 66.kDa.kDa 9.kDa.kDa.kDa Bioseparation products catalog

15 BioPro Ion Exchange Screening Kits. Transferrin (. mg/ml). Trypsin inhibitor (. mg/ml). Ribonuclease A (. mg/ml). Cytochrome c (. mg/ml). Lysozyme (. mg/ml) BioPro Q7 BioPro S7, BioPro DA6 BioPro CM6 DA DB Eluent Temperature Injection : ml type (6 X 7. mmi.d.) : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing.m NaCl -8%B (- ) : 8 cm/hr (.6 ml/) : : UV at nm : L Eluent Temperature Injection : ml type (6 X 7. mmi.d.) : A) mm NaHPO - -NaHPO (ph 6.8) B) mm NaHPO - -NaHPO (ph 6.8) containing. M NaCl -%B (- ) : 8 cm/hr (.6 ml/) : : UV at nm : L Bioseparation products catalog

16 YMC-Pack Diol MW 6 Diol- 6 Diol Diol-. IgM. Thyroglobulin. IgA. Fibrinogen. -Globulin 6. IgG 7. Transferrin 8. HSA (human serum albu) 9. -Antitrypsin. Ovalbu. Carbonic anhydrase. Trypsin inhibitor. Myoglobin. -Lactalbu. Ribonuclease A 6. Cytochrome c MW 9, 67, 9,, 8,, 7, 66,,,,, 7,,,7, Elution volume (ml) : YMC-Pack Diol X 8. mmi.d. Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl :. ml/ Temperature : C : UV at 8 nm. Glutamate dehydrogenase. Lactate dehydrogenase. Enolase. Adenylate kinase. Cytochrome c MW 9,, 67,,, : YMC-Pack Diol X 8. mmi.d. Eluent :. M KHPO-KHPOpH 7. containing. M NaCl :.7 ml/ Temperature : ambient : UV at 8 nm Bioseparation products catalog

17 N866A N866E N866M N867B N866F N866B N866C N866D N866K N866G N866H 6 P88A Albu Transferrin IgG 6 6 Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl :. ml/ Temperature : ambient ( C) : UV at 8 nm Eluent :. M KHPO-KHPO (ph 7.) :.7 ml/ Temperature : ambient ( C) : UV at nm Injection : L Sample : Mouse ascites fluid (6 times dilution with water). Insulin (Bovine). Neurotensin. Angiotensin. Glycine Eluent Temperature MW,7,67, 7 : YMC-Pack Diol-6 X 8. mmi.d. :. M KHPO-KHPO ph 7. containing. M NaCl/ acetonitrile7/ :.7 ml/ : ambient : UV at nm MW Myoglobin. Ribonuclease A. Cytochrome c. InsulinBovine). Insulin B chain 6. -Mating factor 7. Neurotensin 8. Angiotensin 9. CCK-Octapeptide. Bradykinin. Angiotensin. Oxytocin. Angiotensin. Met-Enkephalin. Leu-Enkephalin 6. Tetraglycine 7. Glycine MW 7,,7,,7,96,68,67,96,,6,, Elution volume (ml) Diol-. P-8. P-. P-. P- MW 8, 8,,7,8 MW Diol-6 Diol- Diol- 9 Diol- MW. Pullulan (P-8) 8,. Pullulan (P-) 8,. Pullulan (P-) 86,. Pullulan (P-),. Pullulan (P-) 8, 6. Pullulan (P-),7 7. Pullulan (P-), 8. Pullulan (P-),8 9. Maltopentadecaose (G),8. Maltoundecaose (G),8. Maltoheptaose (G7),. Maltopentaose (G) 8. Maltotriose (G). Maltose (G). Glucose (G) 8 Elution volume (ml) Eluent Temperature : YMC-Pack Diol, X 8. mmi.d. : water :. ml/ : ambient : RI Bioseparation products catalog 6

18 7 Bioseparation products catalog Reversed-phase columns and packing materials

19 6 7 A8B 6 7 AD. BAM-P. [D-Ala,Met ]-Enkephalinamide. -Endorphin. Met-Enkephalin. [D-Ala,Met ]-Enkephalin 6. -Endorphin 7. -Endorphin Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -%B (- ), %B (- ) :. ml/ Temperature : 7 C : UV at nm,) 87),76) 7) 88),899),6) C8, nm C8, nm D, 79.D C8, nm C, nm D 7.D C8, nm 6 8.D. Cytochrome c. Insulin (Bovine). Amyloid -protein. Lysozyme. -Lactalbu 6. Myoglobin Eluent Temperature,),7),),),) 7,) : YMC-Pack m, X.6 mmi.d. : A) water/tfa (/.) B) acetonitrile/tfa (/.) -6%B (- ) :. ml/ : 7 C : UV at nm Bioseparation products catalog 8

20 s/packing Materials Eﬀect of column temperature on separation of peptides and proteins Condition A -8,) MPa (,6 -,7 psi) Condition B,-,7) Peak capacity = 6 ºC MPa (,7 -, psi) Analytes Peak capacity = 67, ºC F8B.8-8. MPa (,9 -,68 psi)... *. F6B.... Peak capacity = * Eluent Peak capacity = MPa (,97 -, psi). Gradient F8A Injection. F6A * MW Condition A. Oxytocin. Leu-Enkephalin. β-endorphin. Insulin. β-lactoglobulin A Condition B. Lysozyme. α-chymotrypsinogen. β-lactoglobulin A System * Peak width ½ () 7,7 6,6,7 8, ,,7 8, : YMC-Triart C8 (.9 μm, nm), X. mmi.d. : A) water/tfa (/.) B) acetonitrile/tfa (/.) - condition A B) acetonitrile/-propanol/tfa (//.) - condition B : -8%B (- ) - condition A -6%B (- ) - condition B :. ml/ : UV at nm : μl ( μg/ml) - condition A μl ( μg/ml) - condition B : Agilent SL PC (peak capacity) = + (gradient time/peak width*) *peak width = W.h average The eﬀect of temperature on separation of peptides and proteins with a variety of molecular weights ) is estimated. The separations at C and 7 C are compared. By increasing column temperature to 7 C, selectivity change is observed, and peaks become sharper, and improved resolution especially for larger molecules is obtained. Generally, larger molecules diﬀuse very slowly compared to small molecules. An elevated temperature can improve eﬃciency and peak shape by lowering mobile phase viscosity and improving mass transfer. Temperature is a simple and eﬀective tool to increase resolution in separation of proteins and peptides. Improv Improvement of resolution by increasing column temperature and coupling of.9 μm columns coupli Peak capacity = 6 ºC.9 μm, X. mmi.d. gradient MPa (6,7-7, psi) 7ºC 6 8 P7A Peak capacity =.9 μm, X. mmi.d. gradient MPa (,-, psi) 9 Coupling of two columns P78A Two coupled.9 μm, X. mmi.d. gradient 9. Peak capacity = MPa (8,-8,9 psi) 9 P77A : YMC-Triart C8 (.9 μm, nm) : A) water/tfa (/.) B) acetonitrile/tfa (/.8) -%B (- ) for a single column -%B (- ) for two coupled columns :. ml/ : UV at nm Injection : μl for a single column μl for two coupled columns Sample : Tryptic digest of Bovine Hemoglobin System : Agilent 9 Eluent 9 Bioseparation products catalog % more peaks can be resolved by increasing the column temperature to 7 C in the separation of tryptic digest of Hemoglobin. The outstanding eﬃciency obtained by a coupling of two mm length of Triart.9 μm columns reduces co-elution of peaks and allows the precise separation in an analysis of complicated samples, such as peptide mapping.

21 8 6 NH N O N H Cytosine HN O O N H Uracil HN O CH O N H Thye NH N N NH N Adenine CMP. -UMP. -GMP. -IMP. -TMP 6. -AMP H N HN O N N NH 8 6 B9E Guanine 6 8 C79D : YMC-Triart C8 ( m, nm) X.6 mmi.d. Eluent : mm KHPO-KHPO (ph 6.9) :. ml/ Temperature: 7 : UV at nm Injection : 8 L (.. mg/ml) : YMC-Triart C8 ( m, nm) X. mmi.d. Eluent : mm CHCOOH-CHCOONH (ph.7) :. ml/ Temperature: : UV at 6 nm Injection : L ( g/ml) F6H Gradient : YMC-Triart C8 ( m, nm) X. mmi.d. :. ml/ : -%B (-8 ) Eluent : A) mm di-n-butylae-acetic acid (ph 6.) B) methanol Temperature: : UV at 69 nm Injection : L ( nmol/ml) Gradient : YMC-Triart C8 (.9 m, nm) X. mmi.d. :.8 ml/ : -%B (-7 ) 6 F9K 7 Impurities mer mer Recovery 6%. 7. Impurities 7.. Recovery % '-CCGCTCGAGCTAAAA AAAGCCTGTGTTACC-' purity>99% Eluent : A) mm DBAA* (ph6.)/methanol (6/) B) mm DBAA* (ph6.)/methanol (/8) -%B (- ) Temperature: ambient : UV at 69 nm Sample : synthetic oligonucleotide ( mol/ml) * di-n-butylae-acetic acid Bioseparation products catalog

22 Preparative Systems BioStream Bioseparation products catalog

23 YMC Pilot columns ECO PLUS Bioseparation products catalog

24 X X Worldwide Availability YMC America, Inc. YMC Europe GmbH YMC India Pvt. Ltd. YMC Korea Co., Ltd. YMC Shanghai Rep. Office YMC Singapore Tradelinks Pte. Ltd. YMC Taiwan Co., Ltd. Distributor YMC Karasuma-Gojo Bldg., 8 Daigo-cho Karasuma Nishiiru Gojo-dori, Shimogyo-ku Kyoto, 6-86, Japan TEL: FAX: sales@ymc.co.jp The above information is as of November, and is subject to change without notice. YMC CO., LTD. All Rights Reserved. K A

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