1. Advances in HPLC Purification of Peptides Future Benefits of In-house Integration Imre Sallay*, Keiji Koyanagi* and Oscar R. Rebolledo** *DAISO CO., LTD., Japan and **DAISO Fine Chem USA, Inc., Torrance, CA

2. Outline The job Silica based stationary phase BASICS: - pore size - particle size - bonding density Pulling the right one from the box The jobs and structure of the API company The trouble The benefits of working in harmony

3. The Job In analytical HPLC you seek INFORMATION. (Identification and / or quantification) In analytical HPLC you want separated peaks. In preparative / process HPLC you seek MATERIAL. (Isolation of a single substance at a certain purity) In preparative / process HPLC you want PRODUCTIVITY. (YIELD, LOADING CAPACITY and PURITY grows at the expense of another.) YIELD PURITY LOADING

4. Silica Physicochemical Properties Particle size (  m) Pore size (nm or A) (or 1 nm = 10 A) Silica surface (place for bonding) 10  m 12 nm or 120 A

5. SP- 120 -10-C4-P SP- 200 -10-C4-P SP- 300 -10-C4-P Column: 6 mm I.D. x 250 mm length; Temperature: 35ºC; Detector: UV 220 nm; Mobile phase: A) CH 3 CN/H 2 O/TFA = 20/80/0.1, B) CH 3 CN/H 2 O/TFA = 60/40/0.1, Linear gradient from A to B in 25 min and hold for 10 min; Flow rate: 1.7 ml/min. Protein standards 1. Ribonuclease A (M.W. 13,700) 2. Cytochrome C (M.W. 12,400) 3. Lysozyme(M.W. 14,300) 4. BSA (M.W. 67,000) 5. Myoglobin (M.W. 18,800) 6. Ovalbumin (M.W. 45,300) The Appropriate PORE SIZE

7. PORE SIZE vs. Surface Area pore size Angstrom10012020030010002000 surface area m 2 /g4503002001002515

8. PORE SIZE vs. Loading Capacity L.C. in 10% ACN/0.5% TFA 70 80 90 100 110 120 130 140 150 160 170 50100150200250 Pore Diameter( Å ) Loading Capacity (mg/ml) SP-200-C8-P SP-200-10-C8-BIO SP-120-10-C8-P SP-120-10-C8-BIO A-120-10-C8 B-100-10-C8 SP-60-10-C8-P

9. PARTICLE SIZE vs. Performance particle size micron 35810152050 theoretical plate plates / meter 160,000100,00070,00050,00036,00025,00012,000 back pressure kg/cm 2 75451910631 5  m 10  m 15  m

10. Bonding Density C% average 17% high 20% low 15% 2 ~ 7 1.5 ~ 8 1.5 ~ 10 average high low Medium density HIGH density LOW density HydrophobicitypH range -ODS-BP -ODS-RPS -ODS-BIO

11. Example for Aqueous RP Phase 100% Aqueous Condition 1st injection 4th injection 5th injection 2nd injection 3rd injection 1st injection 4th injection 5th injection 2nd injection 3rd injection after 5th injection keep initial condition after 5th injection phase collapse [Test Condition] Column size : 4.6mmI.D. x 150mmL, Mobile Phase : 100% H 2 O, Flow Rate : 1.0 mL / min, Temperature : 40ºC, Detection : UV, 254 nm Sample : 1. Cytosine, 2. Uracil, 3. Cytidine, 4. Uridine, 5. Thymine SP-120-5-ODS-RPSSP-120-5-ODS-BP

12. [Test Condition] Column size : 4.6mmI.D. x 150mmL Mobile Phase : CH 3 OH / H 2 O = 80 / 20 Flow Rate : 1.0 mL / min Temperature : 40ºC Detection : UV, 254 nm Sample : 1. Uracil 2. Caffeine 3. Phenol 4. n-Butylbenzene 5. o-Terphenyl 6. n-Amylbenzene 7. Triphenylene Difference in Retention Time SP-120-5-ODS-BP SP-120-5-ODS-RPS SP-120-5-ODS-BIO 1 2 3 4 5 6 7

13. Retention Difference Standards for RP silica test -- SP-120-5-ODS-BP -- SP-120-5-ODS-RPS -- SP-120-5-ODS-BIO -- SP-100-5-ODS-P  -3 fatty acid sample -- SP-100-5-ODS-P -- SP-120-5-ODS-RPS -- SP-120-5-ODS-BP -- SP-120-5-ODS-BIO SP-120-5-ODS-BP SP-120-5-ODS-RPS SP-120-5-ODS-BIO SP-100-5-ODS-P = < < SP-120-5-ODS-BIO SP-120-5-ODS-BP SP-120-5-ODS-RPS SP-100-5-ODS-P < < < Retention time Not always same order!!

14. Pulling the Right… SP-120-10-ODS-RPS The general ODS phase, for natural compounds SP-120-10-ODS-BP Aqueous ODS SP-120-10-ODS-BIO Wide pH range SP-100-10-ODS-P High surface area phase, high loading capacity SP-100-10-ODS-PK Super strong, extra wide pH range

15. In-house Communication in Pharma Step 1: Bench top laboratory work Lead discovery, possibly HTS (small quantities, ng ~ mg). Step 2: Gram laboratory Milligrams or grams of hit compounds Step 3: Kilo lab or PILOT plant Up to kilos for phase and other trials Step 4: Production plant Full scale production

16. The Trouble Step 1: Bench top laboratory work Lead discovery, possibly HTS (small quantities, ng ~ mg). Step 2: Gram laboratory Milligrams or grams of hit compounds Step 3: Kilo lab or PILOT plant Up to kilos for phase and other trials Step 4: Production plant Full scale production

17. Summary – Work in HARMONY Because it is nice to be nice Why spend half of your life with people not liking you Job satisfaction – THE BRAGGING RIGHTS!!!

18. Thank You! Thank you for your attention! Bye-bye! See you next time in your lab!

19. The 5 most abundand proteins in serum Serum Albumin Step 1: First the pore size shall be determined. As SEPARATION takes place where the stationary phase meets the mobile phase, namely on the SURFACE of the silica you want a silica with big enough pores for your analyte to have free access to move in-and out of the pores, use the whole Chromatographically Accessible Surface Area of the stationary phase. Screening for Perfect Phase

20. Step 2: Choose the bonding type. First choice should be an “ordinary” ODS phase, (like the DAISOGEL -ODS-RPS), moving down to shorter alkyl chains to establish still good separation but short elution time balance. Step 3: We recommend to play with differences provided with different bonding types and bonding densities, here special needs shall be considered like harsh pH conditions or other specialties like water- rich mobile phases… Step 4: Simply establish the particle size small enough to provide high resolution but big enough for your HPLC system to cope with the back-pressure. Screening for Perfect Phase