Advances in HPLC Purification of Peptides Future Benefits of In-house Integration

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 YIELD PURITY LOADING 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.)

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

5. The Appropriate PORE SIZE 3 4 1 2 5 6 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 min SP-200-10-C4-P SP-120-10-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) CH3CN/H2O/TFA = 20/80/0.1, B) CH3CN/H2O/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)

6. The Appropriate PORE SIZE

7. PORE SIZE vs. Surface Area

8. PORE SIZE vs. Loading Capacity L.C. in 10% ACN/0.5% TFA 170 160 150 SP-120-10-C8-BIO 140 A-120-10-C8 130 SP-120-10-C8-P B-100-10-C8 120 Loading Capacity (mg/ml) 110 SP-200-10-C8-BIO 100 90 SP-200-C8-P 80 SP-60-10-C8-P 70 50 100 150 200 250 Pore Diameter(Å)

9. PARTICLE SIZE vs. Performance 5 mm 10 mm 15 mm

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

11. Example for Aqueous RP Phase 5th injection 5th injection 4th injection 4th injection 3rd injection 3rd injection 2nd injection 2nd injection 1st injection 1st injection 100% Aqueous Condition SP-120-5-ODS-BP SP-120-5-ODS-RPS [Test Condition] Column size : 4.6mmI.D. x 150mmL, Mobile Phase : 100% H2O, Flow Rate : 1.0 mL / min, Temperature : 40ºC, Detection : UV,254 nm Sample : 1. Cytosine, 2. Uracil, 3. Cytidine, 4. Uridine, 5. Thymine after 5th injection after 5th injection keep initial condition phase collapse

12. Difference in Retention Time 1 7 5 2 6 4 3 SP-120-5-ODS-BIO SP-120-5-ODS-RPS SP-120-5-ODS-BP [Test Condition] Column size : 4.6mmI.D. x 150mmL Mobile Phase : CH3OH / H2O = 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

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 SP-120-5-ODS-BP SP-120-5-ODS-RPS SP-120-5-ODS-BIO SP-100-5-ODS-P < = < w-3 fatty acid sample SP-120-5-ODS-BIO SP-120-5-ODS-BP SP-120-5-ODS-RPS SP-100-5-ODS-P < -- SP-100-5-ODS-P -- SP-120-5-ODS-RPS -- SP-120-5-ODS-BP -- SP-120-5-ODS-BIO < < 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. Screening for Perfect Phase 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.

20. Screening for Perfect Phase 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.