[ Process Purification ]

™™ DAISOGELDAISOGELPacking Materials for Process Purification DAISOGELPacking Material for Process Purification - offered by Waters Waters Corporation and Daiso Co., Ltd., Osaka, , have jointly entered into a collaboration to globally market and supply Daisogel™ bulk packing materials for process chromatography.

By combining the purification expertise of both organizations, pharmaceutical and biopharmaceutical companies’ scaling processes for lab-scale to pilot plant or process-scale manufacturing, can now benefit from an unparalleled level of technical and product support.

Daisogel Packing Materials

. Silica-based packing materials for pharmaceutical and biopharmaceutical chromatographic purifications

. High mass loading and excellent resolution

. Excellent durability with extended acidic and alkaline resistance

. Available in multiple kilograms

Daisogel Analytical and Preparative HPLC Columns Packed by Waters

. Analytical columns for screening and method development of isolation and purification methods

. Preparative columns packed with Waters patented Prep OBD™ Technology for scaling up

[3] DAISOGEL Columns Packed with Waters OBD Technology

Column Performance – Identifying the Problem

Laboratory-scale HPLC purification presents many challenges to the chromatographer. Inconsistency in column-to-column performance and OBD Optimized Bed Density lifetimes often result in lost samples, repeat purification runs and poor scalability from small to larger volume columns. Common HPLC prep High-pressure Slurry Packing Axial-ram Slurry Packing column failures are due to mechanical instabilities, especially voids in the Localized Bed Expansion Near Inlet Localized Compression Near Inlet packed bed. The patented OBD Technology1 combines optimized packing processes with a unique set of column designs ensuring increased lifetime OBD Process and better reproducibility. Optimum Bed Density Throughout When a column is used in a preparative HPLC system, it must remain stable during operation. The bed must be packed sufficiently dense to withstand the compressive fluid forces encountered during use. In the case of analytical column dimensions, the necessary packed bed density can be generally achieved using traditional slurry packing methods. As the diameter of the column increases, it becomes increasingly difficult to reach the bed density required for stable, long-term performance, especially when using small particles. The optimum bed density is Optimized Bed Density is achieved by applying pre-determined dependent upon the specific properties of the chromatographic particles axial force only at the column inlet. and column dimensions.

1US Patent # 7,399,410; UK Patent # GB2408469

OBD Column Design – Improved Mechanical Stability

Waters combines high-pressure slurry packing with a carefully calculated axial compression element localized at the less-dense inlet end of the bed. With careful tuning of the packing process for each particle type and column geometry, the Prep OBD design and process results in predictable, uniform density profiles throughout the column. Established procedures ensure that the column bed is not over-compressed or disrupted during the final column packing process. Excess axial compression applied at the inlet can break particles, build bridges, and lower local bed permeability.

Exploded Prep OBD Column Hardware

The patented OBD column hardware design incorporates chemically inert seals made to prevent leaks and a pair of specially designed filter housings with frits and distributors built to withstand high pressures.

[4] Daiso Co., Ltd.

Daiso Co., Ltd., headquartered in Osaka, Japan, is a technology-oriented chemical Daisogel Packing Material company supporting industries including medicine, agriculture, IT, machinery, Daiso Co., Ltd. is one of the leading silica gel manufacturers for liquid chromatography, using inorganic synthesis technology. Since textiles, and construction through 1992, Daiso has added large-scale manufacturing capability for innovative products. Daisogel media to fully support the process purification laboratories worldwide. Daisogel phases are used for various applications including medicines and pharmaceuticals, agricultural chemicals, Product offering includes: food ingredients, biomolecules, liquid crystals and functional dyes. . Daisogel packing materials Daisogel packing materials have been designed to meet the needs of process purification. These speherical silica-based packing materials . Pharmaceutical intermediates allow for high mass loading and maximum resolution which can result . Fine chemicals in reducing the number of repeated injections. Through high density bonding and endcapping, Daisogel packing materials have shown . Proprietary functional chemicals extended life and durability. These benefits have helped customers in reducing their overall purification costs. . soda products and Chlor-Alkali . synthetic and fabricated resin products Quality

Daiso quality control system is based on ISO 9001 for the silica gel manufacturing plant and has a DMF (Drug Master File) on file www.daiso-co.com with the FDA (Food and Drug Administration) for the Daisogel packing materials.

Starting with the raw materials, the quality of Daisogel packing material is strictly controlled at each manufacturing step. Each Daisogel batch is analyzed for pore size, surface area, pore volume and particle size, along with measurements of the silica gel pH, volatile matter, and metal impurities. Daisogel packing materials are designed to perform with consistent selectivity from lot-to-lot and between particle sizes.

[5]

DAISOGEL DAISOGEL SP-120-ODS-BP . Silica-based 120Å C18 phase Packing Materials . Suitable for hydrophilic compounds separation . Longer lifetime in aqueous eluents

. Different selectivity from ODS-RPS DAISOGEL SP-120-ODS-RPS ODS-BP phases are designed to show extended selectivity for . Silica-based 120Å C phase 18 hydrophilic and polar compounds which are either poorly or not . High coverage and exhaustive endcapping retained on other phases. Typical applications are separations of biomolecules and metabolites such as oligosaccharides, amino ODS-RPS packings featuring maximum surface coverage are the ideal choice for a acids, small peptides, nucleotides and organic acids. They are wide variety of organic compounds. Carefully controlled full endcapping leads to endcapped and show similar selectivity as conventional C18 phases optimal performance with acidic, basic and chelating compounds. when being used for separations of hydrophobic compounds with typical reversed-phase eluents. They also show stable base lines Analysis of Vitamin B and high sensitivity even under neutral pH conditions and without buffer or counter-ion additives. 100 Column: DAISOGEL SP-120-5-ODS-RPS 150 x 6 mm Eluent: 10 mM sodium phosphate (pH 5.0) /CH3CN (85/15) Analysis of Pyridine/Phenol Flow Rate: 1.0 mL/min Detection: UV @ 254 nm No change in selectivity or retention after 100 hours of washing 1 Temperature: 40 °C

Compounds: 0 hours 100 hours 50 2 1. Thiamin hydrochloride (Vitamin B1) mV 2. Riboflavin (Vitamin B2) after 0 hrs 100 hrs Pyridine Kpy 0.704 0.686 Phenol Kph 2.129 2.080 Separation α 3.024 3.032

0 4 4 0 5 10 15 20 25 min

Analysis of Organic Acids Analysis of Antioxidants Showing good retention of ascorbic acid 250 2 Column: DAISOGEL SP-120-5-ODS-RPS 150 x 6 mm 5 Eluent: 30 mM sodium phosphate 3 1 (pH 2.5 with H3PO4) 200 Flow Rate: 1.0 mL/min Column: DAISOGEL SP-120-5-ODS-BP Detection: UV @ 210 nm 150 x 6 mm 1 Temperature: 40 °C 2 Eluent: 0.1 M phosphate buffer (pH 3.1)

150 Flow Rate: 1.0 mL/min Compounds: Detection: ECD 1. Malonic acid Temperature: Ambient mV 2. Nortriptyline 3. Acetic acid 100 Compounds: 4 4. Lactic acid 1. Ascorbic acid 5. Citric acid 2. GSH 6 6. Succinic acid 50

0 0 2.5 5.0 7.5 10 min 0 5 10 min

[6]

DAISOGEL SP-200-C8-P

. Silica-based 200Å C8 phase

. Recommended for highly hydrophobic samples

C8-P packing materials are bonded with octyl groups and are fully endcapped. They are recommended for compounds which are too strongly retained on C18 phases. 200Å material is widely used for larger molecules with higher hydrophobicity.

Selectivity Comparison of Three Daisogel Phases

2 Mobile Phase: 65% MeOH /35% 20 mM SP-120-5-ODS-RPS 1 3 5 K2HPO4 buffer, pH 7.0 Temperature: 23.4 °C 4 6 7 Compounds: 2 1. Uracil 1 3 5 SP-120-5-ODS-BP 4 2. Propranolol 6 7 3. Butylparaben 4. Naphthalene 2 3 5 5. Dipropylphthalate 4 SP-200-5-C8-P 6. Acenaphthene 1 6 7 7. Amitriptyline

DAISOGEL SP-120-P Analysis of Fat-Soluble Vitamins under

. Ultra-high purity 120Å silica Normal Phase Conditions

Column: DAISOGEL SP-120-5-P P phases are based on spherical and totally porous silica gel with metal 3 150 x 6 mm 400 impurities specified to be less than 10 ppm each for Al, Fe, Ti and Zr. Eluent: n-Hexane/CHCl3 (60/40) 2 Flow Rate: 1.0 mL/min Due to its narrow particle size distribution, P series packing material for Detection: UV @ 254 nm Temperature: Ambient preparative chromatography enables higher column efficiencies, lower operating pressure and longer column lifetimes. Compounds: 1. Vitamin A Palmitate Using DAISOGEL, scale-up is guaranteed by using the same production 2. Vitamin K1 mV procedure for all grades. 200 3. Vitamin E 4 4. Vitamin K3 P series packing materials for preparative use are available in 10 µm, 5. Vitamin D 15 µm and 20 µm. High purity 40-60 µm spherical silica is a cost 5 effective alternative for medium and low pressure chromatography. 1

Measurement of Pressure Drop 15 SP-120-20-P 0 SP-120-15-P 0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min SP-120-10-P 12 )

/m 9 pa

6 Preparative grades packing material in the past have had a wide particle distribution. The advantage of P Pressure (M series packing material for preparative chromatography is that it enables higher column efficiencies and 3 lower operating pressure due to its narrow particle size distribution.

0 0 10 20 30 40 Linear Velocity (cm/min) Condition: MeOH 100% (40 °C) [7]

DAISOGEL SP-300-BIO

. Suitable for bulky biomolecules, proteins

. 300Å pore size, narrow particle size distribution, ultra high purity totally spherical silica gel

. High density bonding for extreme performance

. Available types: SP-300-ODS-BIO, SP-300-C4-BIO

The improved high density bonding and full endcapping make it most suitable for general use and to separate or purify high molecular weight compounds, especially proteins. Because of significant improvement in durability, these novel phases exhibit excellent acidic and alkaline resistance. These can be used for an extended period of time under acidic mobile-phase condition and rinsed for regeneration with NaOH containing solvents.

The C4 phase is recommended for compounds too strongly retained on ODS C18 phases.

Excellent Durability in Acidic Environment

Accelerated Acidic Duration Test Condition 100

Mobile Phase: CH3CN/1.0% TFA aqueous (10/90), pH 1.0 Temperature: 70 °C 80 Purge Time: 3 hr DAISOGEL SP-300-C4-BIO Competitor A (300Å, C )

l (%) l 4 ia it Competitor B (300Å, C ) Chromatographic Test Condition 60 4

Mobile Phase: CH3OH/H2O (35/65) n to In to n io

Temperature: 40 °C e nt 40

Detection: UV @ 254 nm te Re

Compounds: 20 1. Uracil 2. Methyl Benzoate 3. Toluene 0 4. Naphthalene 0 3 6 9 12 15 18 21 24 Purge Time (hr)

DAISOGEL SP-300-C4-BIO Competitor A (300Å, C4) Competitor B (300Å, C4)

250 CV Purged 250 CV Purged 250 CV Purged

1 2 1 2 1 2

4 4 4 3 3 3 Initial Initial Initial

0 2 4 6 8 10 12 14 16 18 20 min 0 2 4 6 8 10 12 14 16 18 20 min 0 2 4 6 8 10 12 14 16 18 20 min

[8]

Excellent Durability in Alkaline Environment

Accelerated Alkaline Duration Test Condition 110

Mobile Phase: CH3CN/0.01 NaOH aqueous (10/90), pH 12 Temperature: 25 °C 100 Purge Time: 3 hr l (%) tia i 90 Daisogel SP-300-10-C4-BIO Chromatographic Test Condition 76 hours: 68.8% 80 Mobile Phase: CH3OH/H2O (35/65) to In on ei nt

Temperature: 40 °C te Detection: UV @ 254 nm Re 70

60 Compp(etitor C (300Å,,µ 15 µm,, C4) Compounds: 18 hours: 66.6% 1. Uracil 50 2. Methyl Benzoate 01020304050607080 3. Toluene 4. Naphthalene Purge Time (hr)

DAISOGEL SP-300-10-C4-BIO Competitor C (300Å, 15/20 µm, C4)

After 76 hr (25 cycles) After 18 hr (6 cycles)

After 19 hr (6 cycles)

1

2 Initial 1 2 Initial 4 3 4 3

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 min 0 2 4 6 8 10 12 14 16 18 20 min

[9] Ordering Information

DAISOGEL PRODUCT CHARACTERISTICS

E x c e p t i o n a l batch-to-batch Suitable for reproducibility Dynamic Axial Enhanced Compression columns Narrow particle mechanical size distribution stability

Guide to Interpreting daisogel Product Names

high acidic resistance; suitable for organic compound separation Silica particle shape: SP (spherical) low carbon content; suitable for hydrophilic and polar compound Pore size (Å): 120, 200, 300 separation in up to 100% aqueous eluents for compounds too strongly retained on ODS phases Particle size (µm): 5, 10, 15, 20, 40/60 if no bonded phase is indicated, the product is bare silica

Bonded phase: see table designed for biopharmaceutical applications; novel bonding technique ensures strong resistance against alkalic and acidic conditions

SP- 200- 5- C8- P new variation of the C4 phase; novel bonding technique ensures strong resistance against alkalic and acidic conditions

Base silica purity: P indicates ultra-high purity; Bio indicates ultra-high purity

DAISOGEL Physical Properties

Product Name Pore Size (Å) Particle Size (µm) Pore Volume (mL/g) Surface Area (m2/g) % Carbon Endcapping SP-120-ODS-RPS 120 5, 10, 15, 20, 40/60 1.0 300 17 Yes SP-120-ODS-BP 120 5, 10, 15, 20, 40/60 1.0 300 15 Yes SP-200-C8-P 200 5, 10, 15, 20, 40/60 1.1 200 7 Yes SP-120-P 120 5, 10, 15, 20, 40/60 1.0 300 N/A N/A SP-300-ODS-BIO 300 5, 10, 15, 20 0.9 100 8 Yes SP-300-C4-BIO 300 5, 10, 15, 20 0.9 100 3 Yes

[10] DAISOGEL Packing Materials

SP-120-ODS-RPS SP-120-P

Qty 10 µm 15 µm 20 µm 40/60 µm Qty 10 µm 15 µm 20 µm 40/60 µm 100 g 186005014 186005050 186005086 - 100 g 186005008 186005044 186005080 - 500 g 186005015 186005051 186005087 186005117 500 g 186005009 186005045 186005081 186005111 1 kg 186005016 186005052 186005088 186005118 1 kg 186005010 186005046 186005082 186005112 5 kg 186005017 186005053 186005089 186005119 5 kg 186005011 186005047 186005083 186005113 10 kg 186005018 186005054 186005090 186005120 10 kg 186005012 186005048 186005084 186005114 20 kg 186005019 186005055 186005091 186005121 20 kg 186005013 186005049 186005085 186005115

SP-120-ODS-BP SP-300-ODS-BIO

Qty 10 µm 15 µm 20 µm 40/60 µm Qty 10 µm 15 µm 20 µm 100 g 186005002 186005038 186005074 - 100 g 186004990 186005026 186005062 500 g 186005003 186005039 186005075 186005105 500 g 186004991 186005027 186005063 1 kg 186005004 186005040 186005076 186005106 1 kg 186004992 186005028 186005064 5 kg 186005005 186005041 186005077 186005107 5 kg 186004993 186005029 186005065 10 kg 186005006 186005042 186005078 186005108 10 kg 186004994 186005030 186005066 20 kg 186005007 186005043 186005079 186005109 20 kg 186004995 186005031 186005067

SP-200-C8-P SP-300-C4-BIO

Qty 10 µm 15 µm 20 µm 40/60 µm Qty 10 µm 15 µm 20 µm 100 g 186004996 186005032 186005068 - 100 g 186004984 186005020 186005056 500 g 186004997 186005033 186005069 186005099 500 g 186004985 186005021 186005057 1 kg 186004998 186005034 186005070 186005100 1 kg 186004986 186005022 186005058 5 kg 186004999 186005035 186005071 186005101 5 kg 186004987 186005023 186005059 10 kg 186005000 186005036 186005072 186005102 10 kg 186004988 186005024 186005060 20 kg 186005001 186005037 186005073 186005103 20 kg 186004989 186005025 186005061

Daisogel COLUMNS PACKED BY WATERS

SP-120- SP-120- SP-200- SP-300- SP-300- Particle Size Dimension SP-120-P ODS-RPS ODS-BP C8-P ODS-BIO C4-BIO 5 µm 4.6 x 100 mm 186005201 186005171 186005156 186005186 186005141 186005126 5 µm 4.6 x 150 mm 186005202 186005172 186005157 186005187 186005142 186005127 5 µm 4.6 x 250 mm 186005203 186005173 186005158 186005188 186005143 186005128 10 µm 4.6 x 100 mm 186005204 186005174 186005159 186005189 186005144 186005129 10 µm 4.6 x 150 mm 186005205 186005175 186005160 186005190 186005145 186005130 10 µm 4.6 x 250 mm 186005206 186005176 186005161 186005191 186005146 186005131 10 µm OBD 19 x 100 mm 186005207 186005177 186005162 186005192 186005147 186005132 10 µm OBD 19 x 150 mm 186005208 186005178 186005163 186005193 186005148 186005133 10 µm OBD 19 x 250 mm 186005209 186005179 186005164 186005194 186005149 186005134 10 µm OBD 30 x 100 mm 186005210 186005180 186005165 186005195 186005150 186005135 10 µm OBD 30 x 150 mm 186005211 186005181 186005166 186005196 186005151 186005136 10 µm OBD 30 x 250 mm 186005212 186005182 186005167 186005197 186005152 186005137 10 µm OBD 50 x 100 mm 186005213 186005183 186005168 186005198 186005153 186005138 10 µm OBD 50 x 150 mm 186005214 186005184 186005169 186005199 186005154 186005139 10 µm OBD 50 x 250 mm 186005215 186005185 186005170 186005200 186005155 186005140

For additional Daisogel products, please contact your local Waters sales representative.

[11]

Sales Offices

Austria and European Export The Netherlands 31 76 508 7200 (Central South Eastern Europe, CIS Norway 47 6 384 60 50 and Middle East) 43 1 877 18 07 Poland 48 22 6393000 61 2 9933 1777 Puerto Rico 1 787 747 8445 Belgium 32 2 726 1000 65 6593 7100 55 11 5094-3788 Spain 34 936 009 300 1 800 252 4752 x2205 Sweden 46 8 555 11 500 86 21 6879 5888 Switzerland 41 56 676 70 00 CIS/Russia +497 727 4490/290 9737 886 2 2543 1898 Czech Republic 420 2 617 1 1384 United Kingdom 44 208 238 6100 Denmark 45 46 59 8080 All other countries: Finland 09 5659 6288 Waters Corporation U.S.A. France 33 1 30 48 72 00 1 508 478 2000 1 800 252 4752 Germany 49 6196 400600 www.waters.com 852 2964 1800

Hungary 36 1 350 5086

India and India Subcontinent 91 80 2837 1900

Ireland 353 1 448 1500

Italy 39 02 265 0983

Japan 81 3 3471 7191

Korea 82 2 6300 4800

Mexico 52 55 5524 7636

The quality management system of Waters’ manufacturing facilities © 2010 Waters Corporation. Waters, OBD and The Science of What’s Possible are in Taunton, Massachusetts and Wexford, Ireland complies with the Interna- tional Standard ISO 9001:2000 Quality Management and Quality Assurance trademarks of Waters Corporation. DAISOGEL is a trademark of DAISO CO., LTD. Standards. Waters’ quality management system is periodically audited by the registering body to ensure compliance. 720003321EN May 2010 VW-KK-FP