Purification of medicine with Mitsubishi Chemical s separation medias (DIAION TM /SEPABEADS TM )

Transcription

1 Purification of medicine with Mitsubishi Chemical s separation medias (DIAION TM /SEPABEADS TM ) Mitsubishi Chemical Corporation Separation material Division 2015/9/6

2 Contents Base technology of Chromatography separation Interaction between compounds and separation media Characteristic of synthetic adsorbents Comparison of Synthetic adsorbent and ODS Mitsubishi Chemical s adsorbent (MCIGEL TM, DIAION TM /SEPABEADS TM ) Developing Procedure of chromatographic separation How to separate the target material by HPLC Characteristic of Mitsubishi Chemical s Synthetic adsorbent and sample of separation result with HPLC column Research the most suitable separation condition with industrial grade medias Sample of separation result with industrial grade medias Chromatography system Application example of industrial field Others Pressure drop of Mitsubishi Chemical s Synthetic adsorbent Procedure of packing method for Sepabeads P.2

3 Base technology of Chromatography separation Interaction between compounds and separation media Synthetic adsorbents Ion exchange resins Characteristic of synthetic adsorbents Comparison of Synthetic adsorbent and ODS P.3

4 Interaction between compounds and media Synthetic adsorbents, ODS Hydrophobic interaction Polystyrenic adsorbent Stronge interaction Interaction mechanism Silica gel C4, C18 Hydrophilic interaction Polymethacrylic adsorbent Weak interaction Si (CH 2 ) n -CH 3 O O O C O C O NH 2 COOH H 2 N- CH 2 (CH 2 ) 2 -CH 3 H 2 N H N N H NH 2 COOH π-π interaction (double bond effect) Alkyl effect Hydrogen bond effect P

5 Interaction between compounds and media Ion exchange resin Cation Exchange resin R-SO3 - R-COO - Anion exchange resin R-C(CH3)3 + R-NH2 + SO3 - N(CH3)3 + NH +H 3 N N H NH 2 COOH Hydrophobic interaction NH 2 COO - Ion exchange interaction Cation exchange adsorption Anion exchange adsorption And hydrophobic interaction P.5

6 Characteristic of synthetic adsorbent Spherical high crosslinked polymer particle with porous structure No functional residue (no ion exchange reaction) Having high chemical stability DIAION HP20 Adsorb organic compounds on the surface with hydrophobic or hydrophilic interaction Plastic active carbon Highly porous structure Pore diameter is several tens nano-meter High adsorption capacity Selective adsorption utilizing size exclusion effect P.6

7 Physical characteristics Chemical structure Pore Diameter Synthetic adsorbent No functional group on Styrene body or Methacrylate body 60~100 nm (it is possible to adsorb high molecular weigh materials) ODS (Octa Decyl Silyl) Alkyl group on silica body 6~30 nm hydrophobicity CHP07>CHP20>ODS CMG20 depend on chemical structure Chemical stability Theoretical Number High ( it is possible to be industrial chromatography) Low Low high

8 Comparison of Synthetic adsorbents Styrene body Brominated Styrene body Methacrylate body ODS hydrophobicity interaction π-π bond hydrophobic π-π bond hydrophobic Hydrogen bond hydrophobic Hydrophobic Structure selectivity (flat surface effect) (flat surface and Br effect) (flat surface effect) <adsorption image> High selectivity These two compounds adsorbed onto surface., The interaction strength differ depend on its structure. Octa Decyl group move flexibly, interaction strength of these compounds is similar. Surface of synthetic adsorbents Surface of ODS P.8

9 Difference with ODS ODS: Remaining silanol group interact with molecules strongly and cause peak tailing or low recovery. Synthetic adsorbent have same chemical surface. So, adsorption occur in same way on all surface area, that make stable separation. Synthetic adsorbent ODS Some time remaining silanol of ODS effects unfavorably. Hydrophobic interaction Hydrophobic interaction Silanol interaction P.9

10 Separation medias of Mitsubishi Chemical Synthetic adsorbent SEPABEADS TM DIAION TM series Ion exchange resins and protein separation medias DIAION TM SEPABEADS TM FP series P.10

11 Mitsubishi s Synthetic adsorbent Chemical structure Good point Adsorption ability Synthetic adsorbent of Styrene body HP20,HP20SS,HP21 SP70,SP700,SP825,SP850 CHP20,CHP50 Synthetic adsorbent of bromated Styrene body SP207 Standard type Have interaction of π-π bond adsorb materials to have double strongly High hydrophobicity Can adsorb low hydrophobic materials High density(1.2) strong Very strong Synthetic adsorbent of Methacryle ester body HP2MGL SP2MGS CMG20 Have ability of hydrogen bond can adsorb high hydrophilic material for example carboxylic compound, ester compound amino, amid compound it is possible to be chromatography with normal phase hydrophobic weak Hydrophilic strong P.11

12 Mitsubishi s Synthetic adsorbents list Name SP850 SP825L SP70 SP700 HP21 HP20 SP207 HP2MGL Water content 46-52% 52-62% 55-65% 60-70% 45-55% 55-65% 43-53% 55-65% Particle distribution ( 250 m) 90% 95% % ( 250 m) 90% ( 300 m) Effective size (mm) Uniformity coefficient Mean diameter (mm) Surface area(m 2 /g) (m 2 /ml) Pore volume (ml/g) Mode radius (Angstrom) Cephalosporin C adsorption weight (g/l) <10 Note : pre distribution and CPC adsorption weight is reference value P.12

13 Pore distribution Mitsubishi can tailor resins for targeted molecules with chemical & physical structures, like particle size and pore radius. SEPABEADS SP70 SEPABEADS SP700 SEPABEADS SP825L HP20 and HP2MGL have large pores SEPABEADS SP850 DIAION HP21 DIAION HP20 DIAION HP2MGL Lactum MW ~600 Pore Radius (Angstrom) macrolide MW 500~ 1500 Peptide Protein13 MW 1000~100,000 P.13

14 How to select? Key point: MW and hydrophobicity of target materials Target compound Molecular weight of target compound ~ ~ ~several ten thousands Example Pore size small Surface Area large Pore size middle Surface Area middle Pore size big hydrophobic SP850 SP825 HP21 SP70 SP700 HP20 Aromatic Compound, Aliphatic compound with double bond Lowly hydrophobic SP207 Hydrophilic compounds, like oligosaccharide Highly hydrophobic hydrophilic HP2MGL Compound having COOH, COOR, NHR group P.14

15 Scalability Key point: Easy to scale up We can offer separation media for all R&D stage. MCI GEL MCI GEL DIAION DIAION SEPABEADS SEPABEADS 3~10μm 10~50μm 50~250μm >250μm Organic compound CHP20/C04 CMG20/C04 CHP20/P30 CHP50/P30 CMG20/P30 SP20SS/HP20SS SP2MGS HP21 HP20 HP2MGL Sugar and Amino acid CK Series CA Series CK Series CA Series UBK Series UMA Series SK Series SA Series XtalSpeed TM MabSpeed TM FP-DA13 Protein CQA Series CQK Series ChromSpeed TM P.15

16 Procedure of developing of industrial chromatography process First step Study HPLC condition of MCI GEL column Second step Confirm separation condition with industrial grade media with reference of HPLC data P.16

17 Separation a target compounds with MCI GEL column Study HPLC conditions Reversed phase or Normal phase Organic solvent for eluent ACN, MeOH, EtOH, Hex, Hep, AcOEt, CH2Cl2 ph, buffer Relationship of adsorption amount and buffer ph P.17

18 Study HPLC conditions Investigate ODS separation data The information of ODS chromatogram is useful to study HPLC condition of MCI GEL columns. Elution condition Comparison Synthetic adsorbent and ODS Eluent CHP(styrene body) CMG(methacrylate body) ACN ODS content ±5% Half of ODS content EtOH ODS content+20% Half of ODS content ph Basically, same ph of ODS condition General Flow rate 4.6mm*150mmL ml/min 10mm*250mmL 1.0 ml/min P.18

19 Optimization of ACN conc Acetonitrile eluet - The retention of CHP20/C10 and ODS is almost same. Try separation with same acetonitrile conc CHP10S, AcCN 25% Luna C18 (2), AcCN 25% 1.8E E E E E-01 Mitomycin C 8.0E E E-02 UV 360nm (AU) 1.4E E E E E E-02 Mitomycin A 7.0E E E E E E-03 UV 360nm (AU) 7.0E E E E E E-01 Mitomycin C Mitomycin A 3.5E E E E E E E E E E E E E E E E E E time (min) time (min) Column: CHP10S (10 m, 250 x 4.6mm I.D., ) and Luna C18 (2) (10 m, 250 x 4.6mmI.D., ) Eluent: 25% AcCN; Flow rate: 1.00ml/min; Detection: UV360nm; Temperature: 25 C Samples: Mitomycin C, 1mg/ml and Mitomycin A, 0.33mg/ml; CHP10S = CHP20/C10 Injection: 2 l for Mitomycin C and 15 l for Mitomycin A P.19

20 Opitmization of EtOH conc EtOH eluent - CHP20/C10 is stronger hydrophobicity than ODS in EtOH eluent. Try higher EtOH conc than ODS ( %) CHP10S, EtOH 60% Luna C18 (2), EtOH 30% 3.6E E E E E-01 Mitomycin C Mitomycin A 1.6E E-01 Mitomycin C Mitomycin A 4.0E E E E E-02 UV 360nm (AU) 2.4E E E E E E E E E E-03 UV 360nm (AU) 6.0E E E E E E E E E E E E E E E E E E E E E E time (min) time (min) Column: CHP10S (10 m, 250 x 4.6mm I.D., ) and Luna C18 (2) (10 m, 250 x 4.6mmI.D., ) Eluent: 60% EtOH for CHP10S, 30% EtOH for Luna C18 (2); Flow rate: 0.50ml/min; Detection: UV360nm; Temperature: 25 C Samples: Mitomycin C, 1mg/ml and Mitomycin A, 0.33mg/ml; Injection: 2 l for Mitomycin C and 15 l for Mitomycin A CHP10S = CHP20/C10 P.20

21 ph effect for adsorption Organic acid Organic acid is adsorbed more in low ph in reverse phase mode Organic base Organic base is adsorbed more in high ph in reverse phase mode Amino acid Amino acid of isolate point ph is adsorbed max. in reverse phase media P.21

22 Effect of chemical structure M.W. and adsorption amount Large molecule compounds is adsorbed more strongly Polarity and adsorption amount Polar compounds, having many following functional group is less adsorbed onto styrene polymer. OH, NH2, COOH Compound with double bond is adsorbed strongly on synthetic adsorbent due toπ-π bound Flat compound: it is adsorbed strongly on synthetic adsorbent due to π-π bound P.22

23 Advantages of Synthetic adsorbent High selectivity toward double bond structure Recognition of small chemical structure difference Superior recovery yield for peptide, protein and RNA than ODS: low non-reversible binding Both Normal phase and reversed phase chromatography is available. Alkaline tolerant P.23

24 Polyene antibiotics CHP20/C10 recognized double bond difference; π-π interaction Styrene body CHP10S CHP20/C10 ODS LUNA Luna C18(2), (2) 10 m 10 μm 4.5E E E-01 Amphotericin B E E-01 Nystatin E-01 Filipin E E Absorbance (AU) 2.5E E E UV 340nm (AU) Absorbance (AU) 5.0E E E UV 340nm (AU) 1.0E E E E E E E E time (min) time (min) CHP10S = CHP20/C10 Column: CHP10S (10 m, , 250 x 4.6mm I.D.) and Luna C18(2) (10 m, 250 x 4.6mmI.D., ) Eluent A: 0.1% Formic acid; Eluent B: 0.1% Formic acid in AcCN; A/B=60/40; Flow rate: 1.00ml/min; Detection: UV305nm for Nystatin, VIS405nm for Amphotericin B and UV340nm for Filipin; Temperature: 25 C Samples: Nystatin, Amphotericin B and Filipin, saturated in 50% AcCN each; Injection: 10 l P.24

25 Statins CHP20/C10: recognized small difference of chemical structure. Pravastatin Mevastatin Simvastatin Each statin has its impurities which has small structure difference. 4.5E E E-01 CHP10S CHP20/C10 Luna C18(2), 10 m ODS LUNA (2) 10 μm 9.9E E E-01 UV 238nm (AU) 3.0E E E E E-01 UV 238nm (AU) 6.6E E E E E E E E E E E time (min) time (min) Column: CHP10S (10 m, , 250 x 4.6mm I.D.) and Luna C18(2) (10 m, 250 x 4.6mmI.D., ) Eluent A: 0.1% Formic acid; Eluent B: 0.1% Formic acid in AcCN; Flow rate: 1.00ml/min; Gradient: 45%B - 95%B over 20min Detection: UV238nm; Temperature: 25 C Samples: Pravastatin sodium, Mevastatin and Simvastatin, 1mg/ml each; Injection: 5 l CHP10S = CHP20/C10 P.25

26 Peptide separation (Insulin) Conditions 1 2 YMC-Pack ODS-A Styrene body CHP20/C10 Methacrylate body CMG20/C10 Column : 1 YMC-Pack ODS-A 2 CHP20/C10 3 CMG20/C10 Column size : 150 x 4.6mmI.D. Flow rate : 1.0ml/min Eluent A) : water/tfa = 100/0.1 B) : acetonitrile/tfa = 100/0.1 Gradient : 10-60%B over 25min Sample : 1 Insulin glargine 2 Insulin human recombinant Injection : 10μL (1mg/mL) Temperature : 40 Detection : UV 280nm Polymer media showed superior performance for Insulin separation (min) P.26

27 Peptide Separation (Leuplin) LH-RH(Luteinizing hormone) and similar Methacrylate CMG20/C10 is superior peptide separation than ODS. CMG20/C10 (Polymethacrylate 10 m) ODS (10 m) Leuprorelin LHRH human LHRH salmon Buserelin Peptide having Trp (pi:5.89) is adsorbed Styrene body media strongly. Leuprorelin LHRH human LHRH salmon Buserelin UV 280nm UV 280nm time (min) Conditions: Column size, 150 x 4.6mmI.D.; Flow rate, 1.0ml/min; time (min) Eluent A, 0.1% TFA; Eluent B, 0.1% TFA in AcCN; Gradient, 20-60%B over 20min; Sample concentration, 1mg/ml; Injection, 10 l; Temperature, 40deg-C; Detection, UV 280nm. P.27

28 Chemical structure of Leuprorelin and related peptides LH-RH(Luteinizing hormone and similar) Leuplin is medicine for prostate and breast cancer. Arg (pi:10.8) - Try (pi: 5.9) P.28

29 Peptide separation (Surfactin) C18 modified Methacrylate CHPOD/C04 60%B-100%B over25min (F.R.=0.5mL/min) YMC-Pack ODS-A 75%B-100%B over 25min (BV) 8 Conditions Column : 1 CHPOD/C04 2 YMC-Pack ODS-A Column size : 150 x 4.6mmI.D. Flow rate : 0.5, 1.0ml/min Eluent A) : water/tfa = 100/0.1 B) : acetonitrile/tfa = 100/0.1 Gradient : 1 60%B-100%B over 25min 2 75%B-100%B over 25min Sample : Surfactin Injection : 10μL (1mg/mL) Temperature : 40 Detection : UV 215nm CHPOD/C04 is superior for surfactins separation P.

30 Protein separation (A) CHP10S (10 m) CHP20/C10 10μm (B) YMC-Pack ODS-A S-10 12nm (10 m) ODS 10μm UV 280nm(AU) MW=13.7kDa Ribonuclease A Conalbumin Ribonuclease A Conalbumin MW=76kDa UV 280nm(AU) Peak area deceased. => Protein adsorption in ODS time (min) time (min) Separation of proteins on uniform sized 10 m polystyrenic adsorbent and conventional 10 m ODS. Column size: 150 x 4.6mm I.D.; Flow rate: 1.00ml/min. Eluent A, 0.1% TFA; Eluent B, 0.1% TFA in AcCN Gradient, 20%B - 60%B over 20min; Detection, UV 280nm; Samples, Ribonuclease A and Conalbumin 2mg/ml; Injection, 10 l No reversible adsorption was seen in CHP20/C04

31 Linalyl Acetate Linalyl acetate O O Normal Phase chromatography with CMG20 Conditions Linalol impurity Silica YMC-Pack SIL Column : 1 YMC-Pack SIL 2 CMG20/C10 Column size : 150 x 4.6mmI.D. Flow rate : 1.0ml/min Eluent : Hexane/EtOH = 99.5/0.5 (isocratic) Sample : 1 Linalyl Acetate (1mg/mL) 2 Linalool (1mg/mL) 3 extract from lavender Injection : 10μL Temperature : 40 Detection : UV 210nm Linalol impurity Methacrylate body CMG20/C The retention of Linalol was almost same, but other compounds were slightly different.

32 Double-stranded RNA YMC-Pack ODS-A Styrene body CHP20/C Conditions Column : 1 YMC-Pack ODS-A 2 CHP20/C10 Column size : 150 x 4.6mmI.D. Flow rate : 1.0ml/min Eluent A) : water in 100mM TEAA Eluent B) : acetonitrile in 100mM TEAA Gradient 10%B-40%B over 30min Sample : Luciferase GL-2 sirna Injection : 10μL (20μM) Temperature : 40, 50, 60 Detection : UV 260nm (min) (min) 20 CHP20/C10 have homogeneous surface. Can separate double-stranded RNA better than ODS. P.32

33 Single stranded RNA Conditions YMC-Pack ODS-A Column : 1 YMC-Pack ODS-A B) 8-40% over 30min Peak Area 145,000 2 CHP20/C10 Column size : 150 x 4.6mmI.D. Flow rate : 1.0ml/min Eluent A) : water in 100mM TEAA B) : acetonitrile Styrene body 0.4 times in 100mM TEAA CHP20/C10 Gradient : 1 8%B-40%B over 30min B) 10-40% over 30min 22 Peak Area 370, %B-40%B over 30min 26 Sample ssrna Ladder Marker 30 Injection : 5μL (0.11mg/mL) Temperature : 40 Detection : UV 260nm ssrna Ladder Marker base ssrna amount/2.5μl 14 42ng (10pmol) ng (20pmol) 22 33ng (5pmol) (min) 26 39ng (5pmol) 30 45ng (5pmol) CHP20/C10 : Better separation for ssrna Lower non-specific binding P.33

34 Tocopherol/Tocotrienol - Normal phase chromatography with CMG20/C04 Column : Methacrylate body CMG20/C04 Elutant : Hexane/EtOH Silica シリカ系カラム Methacrylate メタクリル系カラム body CMG20/CO4 3 3 Tocopherol group (2) (6) 8 Tocotrienol group 1α-Tocopherol 2β-Tocopherol 3γ-Tocopherol 4δ-Tocopherol 5α-Tocotrienol 6β-Tocotrienol 7γ-Tocotrienol 8δ-Tocotrienol P.34

35 Developing of industrial chromatography system Chromatography system Relationship of particle size and ability of separation Procedure of procedure for developing of industrial chromatography system Sample real examination Colistin purification Vitamin E purification Select chromatography system P.35

36 System1: Selective adsorption Compounds adsorbed onto synthetic adsorbent and others can be separated. Feed raw materials until leakage of target compound Elute target compound with eluent. 原液 原液 水洗 溶離液 Raw material Raw material Rinsing eluent Target A impurities 原液負荷押出溶離押出 Load Displacement Eluting Displacement Concentration 濃度 -Impurity 不純物 B -Target 目的物 A A 溶離体積 Eluent Volume(BV) P.36

37 System2: Chromatography Separate compound A,B by chromatography Feed small volume of raw materials Elute compound A, B with eluent (organic solvent or buffer solution). Weakly binding compound elute firstly. Raw material 原液 溶離液 溶離液 溶離液 elutant eluent eluent eluent Target A ImpurityB 原液負荷 Load 溶離 eluate Concentration 濃度 -Impurity 不純物 B B -Target 目的物 A A 溶離体積 Elutent Volume(BV) P.37

38 System 3: Step gradient chromatography Change concentration of organic solvent or ph step by step enhance strongly binding compound s elution <Example> Eluent 溶離液溶離液 Eluent Eluent 溶離液 Raw 原 material 料 20%MeOH 40%MeOH 40%MeOH 60%MeOH Note) For compounds with amino residue, or carboxylic acid, ph gradient is useful. Raw material 20%MeOH 40%MeOH 60%MeOH displacment Concentration 濃度 Eluent Volume(BV) 液量 P.38

39 Particle size effect on separation H 2 N Smaller size media shows better separation O H H N S CH 3 CH 3 COONa Aminopenicillanic acid 2 H H CONH N O Penicillin G S CH 3 CH 3 COONa O CONH O H Penicillin V H N S CH 3 CH 3 COONa Separation result of some penicillin compound CHP50/P20 CHP50/P30 CHP20/P50 SP20SS HP20SS (18mm) (30mm) (55mm) (70mm) (100mm) Separation condition Column size, 250 x 10mm I.D.; Eluent, MeOH/50mM phosphate (ph8.0) = 60/40; Flow rate, 2.18ml/min; Detection, UV 254nm. Samples: 1= 6-aminopenicillanic acid (1g/L), 2= penicillin G (1g/L), 3= penicillin V (1g/L). Injection Volume: 100ml P.39

40 R&D Steps for industrial process Developing industrial chromatography system as follow Research separation condition with HPLC column (for example to use Method Scouting system) Select HPLC column Decide elution condition(eluent composition, content, ph) Developing separation condition with Industrial grade media(100~400μm) Decrease theoretical plate to apply Industrial grade media. Therefore, we need to lower floe rate and to be higher column height. Resin height HPLC column 4.6*150mmL industrial 1m*1mL Flow rate HPLC 1ml/min=SV24 Industrial SV1 Decide load so on separation condition for reference HPLC data Decide chromatography system P.40

41 Example of scale up development with chromatography STEP 1 Select media HPLC analysis decide HPL column decide elution condition HPLC column MCI GEL CHP20/C04 CMG20/C04 STEP 2 Preparative Dynamic Axial Compression column Media for preparative MCI GEL CHP20/P30 CMG20/P30 Open column Decide as follow Load Productivity Cost STEP 3 Pilot Industrial grade DIAION HP20 HP20SS HP2MG SPMGS Purity Productivity reproduction life Select process single column SMB P.41

42 Experiment Equipment HPLC Equipment Eluent HPLC pump Easy operation Saving hours Can get many data Get good separation Get easy best elution condition Single column equipment Raw Eluent High load more than HPLC OK to be top of each peak. Can apply industrial chromatography separation system. injecture Time analyze ph, composition Detector Time Sampling(total15~30point) P.42

43 HPLC Industrial grade media Styrene body CHP20/C10(10μm) Styrene body SP20SS (70μm) Colistin B Colistin A Colistin A Colistin B Column: CHP20/C04 4.6mmφ*250mmL Eluent: MeOH/0.2M Phosphate(2.5)=50/50 Flow Rate: 0.46ml/min (SV6.7) Detector: UV210nm Temperature: 25 Sample: Colustin sulfate(sigma) Concentration: 10mg/ml Injection: 20μl Column : φ mm Separation media: SP20SS Eluent : Acetonitolyl/0.05M bbuffer(ph3)=20/80 Flow rate : SV1 Temperature : 50 Sample con. : Colustin Broth 100g/L Load : 0.025BV (2.5g/L-R) P.43

44 Separation with single column Colistin A Colistin B Eliminate Colistin B with single column colistina purity 93.2% recovery colistin A Column : φ mm Separation media: SP20SS Eluent : Acetonitolyl/0.05M bbuffer(ph3)=20/80 Flow rate : SV1 Temperature : 50 Sample con. : Colustin Broth 100g/L Load : 0.025BV (2.5g/L-R) P.44

45 Separation with SMB C18 modified Methacrylate body (protocol) Purify colistin A with MSB Target A B C D E Broth purity 58.9% 21.1% 6.0% 1.6% 1.8% crudely made purity 93.2% 0.00% 1.6% 3.2% 0.92% final made purity 99.69% 0.00% 0.00% 0.00% 0.00% recovery 95% Column : φ30 500mm Separation media: New product Eluent : Acetonitolyl/0.05M buffer(ph2)=18/82 Flow rate : SV1 Temperature : 50 Sample con. : Cryde sample 25g/L Load : 0.017BV (0.42g/L-R) P.45

46 HPLC industrial grade Normal phase chromatography Tocopherol - 1 (2) (6) Methacrylate body CMG20/C [min] 8 Methacrylate body SPMG40/C04 Conditions Column : CMG20/C04(4μm) SPMGSS(140μm) Column size 4.5 :150 x 4.6mmI.D. 42.1mmφ*550mmL Flow rate : 1.0ml/min Eluent : Hexane/EtOH = 98/2 Sample : トコフェロール トコトリエノール混合液 1 α-tocopherol 2 β-tocopherol 3 γ-tocopherol 4 δ-tocopherol 5 α-tocotrienol 6 β-tocotrienol 7 γ-tocotrienol 8 δ-tocotrienol Injection : 10μL ( 1mg/mL ) Temperature : 25 Detection : UV 292nm Tocopherol Tocotrienol P.46

47 Separation of Tocopherol with SMB Tocopherol group 7 5 Tocotorienolgroup Conditions Column : SEPABEADS SP2MGS Column size : mmI.D, 4 Eluent : Hexane/EtOH = 90/10 Process : Improve Simulated moving Bed (ISMB) Sample : トコフェロール トコトリエノール混合液 Load : (1/hr) 261mL/hr SV : 1.0 (1/hr) 2.93L/hr E/F : 4.0 (v/v) P/R : 0.89 (v/v) Temperature : 25 時間 (min) Result of SMB separation Crude purity purity recovery Total Tocopherol 38.4% 99.4% 98.4% Total Tocotorienol 66.7% 97.5% 99.6%

48 Select chromatography system Select chromatography system (single column or SMB) to consider separation result of industrial grade Synthetic Adsorbent. Case of good separation or many impurity Single column chromatography system Case of bad separation and 1 or 3 impurity Simulated Moving Bed chromatography system P.48

49 Industrial purification examples Example of Industrial purification system with Synthetic adsorbent Cephalosporin C Vancomycin Daptomycin Taclorimus Streptomycin P.49

50 Cephalosporin C purification Displace (DI water) Elute 0.05M NaHCO3 or 10% 2-propanol Displace (DI water) elute 0.07M NaHCO3 or 30% 2-propanol Fermentation Broth Filtration Synthetic adsorbent HP20 Synthetic adsorbent SP825L CPC concentration 10~12g/L ph=2~3 Eliminate protein Purify CPC Weakly anion exchange resin WA30 RO concentration Crystallization P.50

51 Cephalosporin C purification / SP825L Cephalosporin C Impurity Deacetylcephalosporin C ph Purity 90% Recovery 93% Displace impurity with DI water Raise ph and elute CPC Raw material Displacement 0.07M sodium acetate Adsorb CPC at ph2.8 Raw material :CPC 9.968g/L ph=2.8 CPC Load :35g/L-R Flow rate :SV=1.0 Tenperature :5 Volume Feed Volume: 3.51BV Displacement:: DI water 1.5BV Elutant : 0.07M Sodium Acetate 2.5BV Displacement:: DI water 3.5BV Elutan 2 :1%NaOH 0.1BV Displacement:: DI water 0.9BV P.51

52 Vancomycin purification Fermentation Vancomycin concentration 14g/L ph=2~3 Filtration Adjust ph=7~8 with N-NaOH Vancomycin Elutant 5~15%EtOH Elutant 2-Propanol SP825L Strongly cation exchange resin SK1B Weakly anion exchange resin WA30 Active carbon Active Alumina Recommend process for high purity product Elutant 2-Propanol Synthetic Adsorbent HP20SS Crystallization Crystallization P.52

53 Daptomycin purification Displace with 30mM NaCl Elutant with 500mM NaCl Displace with 30mM NaCl Elutant with 500mM NaCl Fermentation Adjust ph Weakly anion exchange resin FP-DA13 Synthetic Adsorbent HP20SS β-daptomycin isomer +Un-hydrodaptomycin 10ppm n-decanic acid ph=6.5 30mM NaCl Displace with 30% AcCN Elute with ph=4.5~5.0 35% AcCN or 45% 2-propanol Weakly anion exchange resin FP-DA13 Daptomycin RO concentration Freezing dry P.53

54 Taclolimus purification Fermentation Elute with 50% acetone Synthetic adsorbent HP20SS AgNO3 in acetone Tacrolimus, ascomycin Compound A Ag Silica Ag type strongly cation exchange resin Tacrolimus P.54

55 Streptomycin purification Fermentation filtration ph=7.5~8.0 Elute with 1N-H 2 SO 4 Weakly cation exchange resin WK40L concentration Active Carbon Crystallizatin P.55

56 Others Pressure drop for Synthetic adsorption Procedure of column packing HP20SS P.56

57 Pressure drop data 1 P.57

58 Pressure drop data 2 P.58

59 Procedure of column packing with 100μm Synthetic adsorbent Packing procedure of Diaion HP20SS, Sepabeadz SP20SS Column size: 20mmφ*500mmH Resin Volume:157mL 1. Put SP20SS, HP20SS 200mL into methanol solution in beaker for that resin swell. 2. Replace air in equipment to methanol. 3. Transfer SP20SS, HP20SS 200mL into slurry reserve by methanol. 4. Brim methanol in slurry reserve and settling 1hrs it. 5. Feed 50% methanol to slurry reviser and column on sires. At this time, Flow rate change from 1ml/min to 50ml/min in 4 hrs. note: If Solvent is used by IPA, Acetone,. Solvent concentration take 15% 6. After flow rate come to 50ml/hr, in addition, continue to feed methanol for 4hrs. 7. After finished to feed methanol, get off slurry reviser from column. Set Fitting to column top. 8. Replace from 50% methanol to eluent. <Packing equipment> P.59