Peptide Purification Method Development: Application for the Purification of Bivalirudin Marc Jacob, Joshua Heng, and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 91 USA In this technical application, we present the purification method development and scale up experiments performed on a crude sample of Bivalirudin prepared by solid phase synthesis. Introduction The Active Pharmaceutical Ingredient (API), Bivalirudin shown in Figure 1 is a ao acid peptide containing one basic and ao acid residues. With a chemical formula of C 98 H 138 N 24 O 33 and a molecular weight of 218.3 g/ mol, Bivalirudin is also known as Angiomax. It is a direct thrombin inhibitor indicated for use as an anticoagulant. We screened the following silica-based stationary phases: Luna C, Luna C8(2), Luna C18(2), Luna Phenyl-Hexyl, Gei C18, and Synergi Polar-RP, as well as, the following buffers: trifluoroacetic acid (TFA), triethyl ammonium phosphate (TEAP), ammonium acetate, and potassium phosphate. The result of the phase screening, buffer used, and the scale up experiment for the best tested condition are presented. Several reversed phase (RP) sorbents were tested in combination with various chromatographic conditions for the purification of Bivalirudin, a fairly hydrophilic peptide that elutes at 27 % of Acetonitrile on a C18 bonded sorbent. The crude peptide was synthesized by solid phase peptide synthesis (SPPS) and after TFA cleavage the crude peptide was obtained with a crude purity of 71.3 % as shown in Figure 2. The sample was produced by CS Bio, Co. and the synthesis was not optimized. 4 4 Figure 2. Bivalirudin Crude Purity 1 2 Peak No. Time () Area Area % 1 14.1 47 9.7 2 14.87 3611 71.3 3.3 62 13.1 4.9 8.9 Column: Luna µm C8(2) Dimensions: x 4.6 mm Part No.: G-4249-E Mobile Phase A:.1 % TFA in Water B:.1 % TFA in Acetonitrile Gradient: -6 % B in 4, hold 8 % B for Flow Rate: 1 ml/ Temperature: C Detection: UV @ 2 nm Injection Volume: μl Sample Concentration: 1. mg/ml in water 3 4 App ID 19726 Figure 1. Bivalirudin Structure H-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH Page 1 of 8
Material and Methods All chemicals used in this study were purchased from Sigma- Aldrich (St. Louis, MO, USA). All solvents were purchased from EMD (San Diego, CA, USA). The crude bivalirudin sample was kindly provided by CS Bio, Co. (Menlo Park, CA, USA). All HPLC columns were obtained from Phenomenex (Torrance, CA, USA). All chromatography were performed on an Agilent 1 HPLC system from Agilent Technologies (Santa Clara, CA, USA) equipped with a quaternary pump, degasser, variable UV wavelength detector and autosampler. The mobile phase additives tested for in this purification development study were TFA, TEAP, potassium phosphate, and ammonium acetate respectively presented in Figures 3, 4,, and 6. Each chromatogram represented is an enlargement of the region of interest. The results suggest that the best separation is achieved on Luna C8(2) and Luna C18(2) using ammonium acetate ph 4.7 or potassium phosphate ph 7 as mobile phase additives as depicted respectively in Figures and 6. Results and Discussion Screening Experiments Based on the peptide molecular weight and hydrophilicity, the following sorbents were tested for selectivity: Luna C, Luna C8(2), Luna C18(2), Luna Phenyl-Hexyl, Gei C18, and Synergi Polar-RP. For screening purposes, sorbents with particle size of µm and 4 µm were chosen. Using a column selector, we were able to evaluate 6 sorbents with a variety of mobile phases in less than 24 hours. Peptide purification is a complex process where a number of chemical additives can be added to the mobile phases in order to improve the separation of impurities from the targeted product. In Table 1 are listed some of the most common mobile phase additives used in peptide purification as well as the recommended operational ph. Table 1. Mobile Phase Additive and ph for Peptide Purification Compound Formula pk a Recommended ph TFA CF 3 CO 2 H.3 1.-2.3 MSA* CH 3 SO 3 H -1.9 1.-2.2 Formic acid HCO 2 H 3.8 2.8-4.8 Acetic acid CH 3 CO 2 H 4.8 3.8-.8 TEAP (Phosphate 1) TEAH 2 PO 4 2.2 1.-3.2 TEAP (Phosphate 2) (TEA) 2 HPO 4 7.2 6.2-8.2 Potassium phosphate KH 2 PO 4 7.2 6.2-8.2 Ammonia NH 4 OH 9.2 8.2-9. Ammonium acetate NH 4 CH 3 CO 2 9.2 6.-9. Ammonium bicarbonate NH 4 HCO 3 9.2 7.8-9. Ammonium formate NH 4 HCO 3 9.2 7.8-9. *MSA = methanesulfonic acid Page 2 of 8
Figure 3..1% TFA Screening Results 4 4 Luna µm C Luna µm Phenyl-Hexyl 36 12 13 14 16 17 18 19 21 12 14 16 17 18 19 21 4 4 4 Luna µm C8(2) 4 Gei µm C18 12 13 14 16 17 18 19 21 11 12 13 14 16 17 18 19 4 4 Luna µm C18(2) 4 Synergi 4 µm Polar RP 4 12 13 14 16 17 18 19 21 16 17 18 19 21 22 23 24 Column: As noted Dimensions: x 4.6 mm Mobile Phase: A:.1% TFA in Water B:.1% TFA in Acetonitrile Gradient: -6 % B in 4 (except for Synergi Polar-RP with - % B in 4 ) to 8 % in Flow Rate: 1 ml/ Temperature: C Detection: UV @ 2 nm Injection Volume: μl Sample Concentration: 1. mg/ml in water Page 3 of 8
Figure 4. mm TEAP ph 2.3 Screening Results Luna µm C Luna µm Phenyl-Hexyl Luna µm C8(2) Gei µm C18 Luna µm C18(2) Synergi 4 µm Polar-RP Column: As noted Dimensions: x 4.6 mm Mobile Phase: A: ml of Acetonitrile in 1 ml of mm (TEAP) ph 2.3 B: 9 ml of Acetonitrile in 6 ml of mm (TEAP) ph 2.3 Gradient: -9 % B in 4 Flow Rate: 1 ml/ Temperature: C Detection: UV @ 2 nm Injection Volume: μl Sample Concentration: 1. mg/ml in water Page 4 of 8
Figure. mm Potassium Phosphate ph 7 Results Figure 6. mm Ammonium Acetate ph 4.7 Results 12.39 Area: 8.79 Luna µm C8(2) Luna µm C8(2) - 9 9.. 11 11. 12 12. 13 13. 11.2-9 9.. 11 11. 12 12. 13 13. Luna µm C18(2) Luna µm C18(2) 12.39 Area: 8.79-9 9.. 11 11. 12 12. 13 13. Column: As noted Dimensions: x 4.6 mm Mobile Phase A: mm Potassium phosphate ph 7 in Water B:.1 % TFA in Acetonitrile Gradient: -4 % B in 4 Flow Rate: 1 ml/ Temperature: C Detection: UV @ 2 nm Injection Volume: μl Sample Concentration: 1. mg/ml in water Column: As noted Dimensions: x 4.6 mm Mobile Phase: A: mm Ammonium acetate ph 4.7 in Water/Acetonitrile (1:9) B: mm Ammonium acetate in Water/Acetonitrile (1:1) Gradient: to % B in 4 Flow Rate: 1 ml/ Temperature: C Detection: UV @ 2 nm Injection Volume: μl Sample Concentration: 1. mg/ml in water Page of 8
Overload Experiments The targeted purity for the final product was an overall purity greater than 98. % with no single impurity greater than 1. %. Based on the preliary screening experiments, overload experiments were performed on Luna C8(2) Å µm-prep x 4.6 mm column. For purification development, it is best to use the same column length and particle size than the ones intended for scale up to a large column. The elution profile for the overload experiment using ammonium acetate at ph 4.7 is shown in Figure 7. The purification fractions collected were analyzed on a Luna C8(2) µm x 4.6 mm column and the results are shown in Table 2. Figure 7. Purification Elution Profile at 1. % Specific Load 4 4 Column: Luna µm-prep C8(2) Dimensions: x 4.6 mm Part No.: G-4324-E Mobile Phase: A: mm Ammonium acetate ph 4.7 in Water B: Acetonitrile Gradient: to % B in 4 ; hold at 8 % B and % C for ; re-equilibration at 9 % A and % for Flow Rate: 1 ml/ Temperature: C Detection: UV @ 28 nm Injection Volume: ml Sample Concentration: 7 mg/ml in water Area Table 2. Purification - Fraction Analysis RT () Front Impurities (Area) Target Compound (Area) Rear Impurities (Area) 27.4 781 296 27.6 247 98 122 27.8 7 2397 28. 9 412 28.2 89 28.4 7619 39 28.6 917 39 28.8 13 74 29. 1267 224 29.2 1 1469 238 29.4 1 163 24 29.6 183 18191 8 29.8 19 1999 114. 37 293 2339.2 41 9379 448 Analytical HPLC Conditions: Same conditions as Figure 1 using Luna µm C8(2) x 4.6 mm column Front Impurities Target Rear Impurities 27.2 27.4 27.6 27.8 28. 28.2 28.4 28.6 28.8 29. 29.2 29.4 29.6 29.8..2.4.6.8 31. Fraction Collection Time () PhenoLogix: Your Analytical Support Laboratory PhenoLogix is a full-service analytical support laboratory within Phenomenex that specializes in method development and optimization for HPLC, UHPLC, GC, GC/MS, LC/MS/MS, and SPE. Our team of highly experienced scientists provides customized solutions to help streamline your development efforts. PhenoLogix is committed to making the experience of working together easy, effortless, and rewarding. Get started today, and let us exceed your expectations. www.phenomenex.com/phenologix Page 6 of 8
Fractions with purity > 96 % were combined and pooled to recover 8 % of the peptide loaded with an overall purity of 98.3 % with the greatest single impurity at.93 % as demonstrated in Figure 8. Similar overloading experiments were performed using potassium phosphate producing lower recovery for the same purity criteria (data not shown). Figure 8. Purification Recovery and Final Product Purity mau 9 8 7 6 4 11 1 2 12 13 14 18 3 4 Conclusions Crude Bivalirudin obtained by SPPS with a crude purity of 71.3 % was purified by reversed phase chromatography using Luna C8(2) Å µm-prep as sorbent. The best recovery and throughput was achieved by using ammonium acetate at ph 4.7 as a mobile phase which allowed us to prepare a Bivalirudin sample with purity greater than 98. % and a recovery of 8 % in a single stage purification. Additionally, it is important to point out that the final product was prepared as an acetate salt form which most of the time is the required salt form used as a peptide pharmaceutical ingredient. 11 Combined fractions 27.8 29.8 ; Recovery 8. % with purity 98. % Peak No. Time () Area Area % 1 12.74 73.7. 2 13.83 4.6.19 3 14.37 21118.7 98.3 4.88..93 Ordering Information Scout Columns (mm) Luna ( Å) Phases x 4.6 x μm-prep C18(2) G-4324-E G-4324-N C8(2) G-4323-E G-4323-N C4(2) G-446-E G-446-N Phenyl-Hexyl G-43-E G-43-N Silica(2) G-4322-E G-4322-N μm C18(2) G-4273-E G-4273-N C8(2) G-4272-E G-4272-N Phenyl-Hexyl G-4286-E G-4286-N Silica(2) G-4271-E G-4271-N Bulk Media Luna ( Å) Phases g 1 kg kg kg kg kg μm-prep C18(2) 4G-4324 4K-4324 4L-4324 4M-4324 4N-4324 4P-4324 C8(2) 4G-4323 4K-4323 4L-4323 4M-4323 4N-4323 4P-4323 C4(2) 4G-446 4K-446 4L-446 4M-446 4N-446 4P-446 Phenyl-Hexyl 4G-43 4K-43 4L-43 4M-43 4N-43 4P-43 Silica(2) 4G-4322 4K-4322 4L-4322 4M-4322 4N-4322 4P-4322 μm C18(2) 4G-4273 4K-4273 4L-4273 4M-4273 4N-4273 4P-4273 C8(2) 4G-4272 4K-4272 4L-4272 4M-4272 4N-4272 4P-4272 Phenyl-Hexyl 4G-4286 4K-4286 4L-4286 4M-4286 4N-4286 4P-4286 Silica(2) 4G-4271 4K-4271 4L-4271 4M-4271 4N-4271 4P-4271 Synergi (8 Å) Phases x 4.6 x μm Polar-RP G-41-E G-41-N Synergi (8 Å) Phases g 1 kg kg kg kg kg μm Polar-RP 4G-41 4K-41 4L-41 4M-41 4N-41 4P-41 Gei (1 Å) Phases x 4.6 x μm C18 G-4436-E G-4436-N Gei (1 Å) Phases g 1 kg kg kg μm C18 4G-4436 4K-4436 4L-4436 4M-4436 Page 7 of 8
Ordering Information Axia Packed Preparative HPLC Columns (mm) Luna ( Å) Brand Phase x 21.2 x x Luna µm C G-492-P-AX G-492-U-AX G-492-V-AX Luna µm PREP C18(2) G-4324-P-AX G-4324-U-AX G-4324-V-AX Luna µm PREP C8(2) G-4323-P-AX Inquire G-4323-V-AX Luna µm PREP Phenyl-Hexyl G-43-P-AX Inquire Inquire Synergi µm Polar-RP G-41-P-AX G-41-U-AX G-41-V-AX Gei µm C18 G-4436-P-AX G-4436-U-AX G-4436-V-AX Australia t: 2-9428-6444 f: 2-9428-644 auinfo@phenomenex.com Austria t: 1-319-11 f: 1-319- anfrage@phenomenex.com Belgium t: 2 3 4 (French) t: 2 11 8666 (Dutch) f: +31 ()-2383749 beinfo@phenomenex.com Canada t: (8) 43-3681 f: (3) 328-7768 info@phenomenex.com Denmark t: 4824 848 f: +4 48 626 nordicinfo@phenomenex.com Finland t: 9 4789 63 f: +4 48 626 nordicinfo@phenomenex.com France t: 1 9 21 f: 1 9 21 11 franceinfo@phenomenex.com Germany t: 621-88- f: 621-88-11 anfrage@phenomenex.com India t: 4-12 24 f: 4-12 2411 indiainfo@phenomenex.com Ireland t: 1 247 4 f: +44 16-1796 eireinfo@phenomenex.com Italy t: 1 632711 f: 1 6327 italiainfo@phenomenex.com Luxembourg t: +31 ()-24187 f: +31 ()-2383749 nlinfo@phenomenex.com Mexico t: 1-8-844-226 f: 1-3-328-7768 tecnicomx@phenomenex.com The Netherlands t: -24187 f: -2383749 nlinfo@phenomenex.com New Zealand t: 9-47891 f: 9-47892 nzinfo@phenomenex.com Norway t: 8 2 f: +4 48 626 nordicinfo@phenomenex.com Puerto Rico t: (8) 41-HPLC f: (3) 328-7768 info@phenomenex.com Sweden t: 8 611 69 f: +4 48 626 nordicinfo@phenomenex.com United Kingdom t: 16-1367 f: 16-1796 ukinfo@phenomenex.com United States t: (3) 212- f: (3) 328-7768 info@phenomenex.com All other countries: Corporate Office USA t: (3) 212- f: (3) 328-7768 info@phenomenex.com If Phenomenex products in this technical note do not provide at least an equivalent separation as compared to other products of the same phase and dimensions, return the product with comparative data within 4 days for a FULL REFUND. Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/termsandconditions. Trademarks Luna and Gei are registered trademarks, and Synergi and Axia are trademarks of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc. www.phenomenex.com Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at international@phenomenex.com Disclaimer Phenomenex is in no way affiliated with Agilent. Comparative separations may not be representative of all applications. Gei is patented by Phenomenex. U.S. Patent No. 7, 674, 383 Axia is patented by Phenomenex. U.S. Patent No. 7, 63, 367 12 Phenomenex, Inc. All rights reserved. TN9418412_W Page 8 of 8