High Throughput Methods for the Identity Tests of Low Molecular Mass Heparins and Heparin Sodium

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2 High Throughput Methods for the Identity Tests of Low Molecular Mass Heparins and Heparin Sodium Transfer of Pharmacopoeial GPC/SEC Analytical Methods to UHPLC Technology F. Spelta, L. Liverani, A. Peluso - OPOCRIN SpA International Symposium on GPC/SEC and Related Techniques October, 1 st Frankfurt 2

3 Overview Heparin & LMM-Hs Regulatory Overview HP-SEC & UHP-SEC; methods transfer Ph.Eur. method transfer & Results USP Enoxaparin method transfer & Results USP Heparin Sodium method transfer & Results USP <209>, draft, LMM-Hs method transfer & Results Conclusions 3

4 Heparin and Low Molecular Mass Heparins Heparin and Low-Molecular-Mass Heparins (LMM- Hs) are the most widely anticoagulants used to prevent and treat thrombosis in patients LMM-Hs are a very important class of semi-synthetic drugs obtained by partial depolymerization of unfractionated Heparin 4

5 Heparin Sources and Structure Heparin is extracted from animal tissues (mainly porcine mucosa) Heparin is a linear, sulfated, polysaccharide. Chains are made up of repeated disaccharide units, each one made up of a uronic acid linked to a glucosamine Most of the free positions can be sulfated, giving a high negative charge to the chain 5

6 Molecular Mass of Heparin and LMM-Hs Heparin has a broad distribution of chains, with a weight-average relative molecular mass (Mw) ranging between 15 and 20 kda LMM-Hs are a class of several products, each one with a characteristic Mw and MM distribution: the Mw of the different products ranges between 3.6 and 7.5 kda 6

7 Molecular Mass of Heparin and LMM-Hs The biological activities and the pharmacokinetics of these products, and therefore the use of these drugs, depend almost exclusively on their Mw and MM distribution Due to the importance and wide availability on the world market of these drugs, the major Regulatory Agencies have defined their quality attributes with general and specific monographs in Pharmacopoeias 7

8 Pharmacopoeias Pharmacopoeias are collections of reference texts published by Regulatory Agencies defining the quality of medicinal products by the standardization of their properties and methods of analysis The major Pharmacopeias in the world are the European (Ph.Eur.), the United States (USP) and the Japanese (JP) In the Ph.Eur., a general monograph (0828) recommends a HP-SEC method for the determination of Mw and distibution of all LMM-Hs 8

9 Pharmacopoeias 5 specific monographs of Ph.Eur. define the specification limits of Mw and MM distribution for as many, different, LMM-Hs In the USP, a specific monograph for just one LMM-H (Enoxaparin) and the monograph for Heparin recommend two different HP-SEC methods The USP is going to issue a new general monograph with a HP-SEC method for all other LMM-Hs 9

10 Ph.Eur. and USP HP-SEC methods characteristics Silica based columns, with specific fractionation ranges, single or coupled, are recommended. Tosoh TSK SW.XL columns are suggested as suitable Recommended mobile phases are Ammonium Acetate, LiNO 3 or Na 2 SO 4 solutions, M Flow rates 0.5 or 0.6 ml/min Run times from 30 min (single column) to 60 min (coupled columns) 10

11 UHPLC for the SEC First UHPLC available in 2004 from Waters (UPLC ) no TSK SW.XL-like columns were available for UHPLC up to 2010 In 2013 Waters completed the BEH series (450, 200 and 125), as UHP-SEC columns for protein analysis In summer 2013 Waters brought a new Acquity UPLC Refractive Index detector on the market 11

12 Method Transfer Early in 2014 in collaboration with Waters-Italy, the first tests on a Waters UPLC equipped with a SEC column BEH200 for Parnaparin, the Opocrin LMM-H After the first results, tests of the other BEH SEC columns for the transfer of all Eur.Ph. and USP methods Method transfer carried out for: i. All LMM-Hs, Identification C - Ph.Eur.; 0828 ii. Enoxaparin Identification D - USP monograph iii. Sodium Heparin Identification D - USP monograph iv. LMM-Hs, MM determination - USP; <209>, draft 12

13 Ph.Eur. general monograph 0828 for LMM-Hs Identification C Column: single, silica based, fractionation range k for proteins, 7.8 x 300 mm (Tosoh TSK G2000 SW.XL is suitable ) Mobile phase: 0.2 M sodium sulphate, ph 5.0 Flow rate: 0.5 ml/min Injection: 25µL Test and Reference solutions: 10 mg/ml Detection: Refractive Index. RI and UV (234nm) only for calibration Run time: not specified (at least 30 min) Calibration: through a complicated procedure, using one moment of a Reference Standard (Mn), and both chromatograms from RI and UV 13

14 Eur.Ph. specifications from the specific monographs of the 5 LMM-Hs, Identification C LMM-Heparin Enoxaparin Nadroparin Parnaparin Dalteparin Tinzaparin Mw ( kda ) < NMT NMT 10 Percentage of chains with MM (kda) < NMT 30 NMT >

15 Ph.Eur LMM-H Identification C Operating conditions Column(s) HPLC TSK G2000SW.XL (5µm) 7.8x300 mm UHPLC Mobile phase 0.2M sodium sulphate, ph 5.0 the same Flow, ml/min Injection Volume, µl Sample / Std conc., mg/ml 10 the same Std LMM-CRS Batch 3 the same RUN TIME 30 min. 6 min. WATERS BEH x150 mm 1.7µm 15

16 Method transfer: Ph.Eur LMM-Hs Identification C MV LMM-H CRS, HPLC Minutes LMM-H CRS, UHPLC µriu LMW_H Minutes 16

17 Method transfer: Ph.Eur LMM-Hs Identification C Comparison between HPLC and UHPLC Six determinations in conditions of Intermediate Precision of the same 5 LMM-Hs, both in HPLC and UHPLC Comparison of Mw and dispersion Comparison with two different values of the calibration moment Cal Mn (3800-official [UPLC 1] and 3900-tentative [UPLC 2] ) for metrological continuity 17

18 Method transfer: Ph.Eur LMM-Hs Identification C Percentage of chains with MM (k) Comparison between HPLC and UHPLC: mean of results from the Intermediate Precision sets LMM-H Enoxaparin Nadroparin Parnaparin System HPLC UPLC 1 UPLC 2 HPLC UPLC 1 UPLC 2 HPLC UPLC 1 UPLC 2 Mw 4390 (0.26) 17.6 < 2.0 < (1.95) 72.2 (0.37) 4310 (0.71) 16.9 (2.37) 73.2 (0.86) 4420 (0.63) 15.7 (2.51) 73.7 (0.83) 4680 (0.29) 7.1 (6.02) 41.7 (0.74) 83.0 (0.46) 4550 (0.48) 6.6 (5.37) 45.6 (2.16) 84.0 (0.51) 4670 (0.51) 5.9 (4.97) 44.2 (2.02) 84.0 (0.51) 5480 (0.29) 27.7 (0.62) 52.8 (0.33) 5350 (0.65) 29.1 (1.09) 52.1 (0.56) 5490 (0.63) 27.8 (0.97) 52.3 (0.46) 18

19 Method transfer: Ph.Eur LMM-Hs Identification C Percentage of chains with MM (k) Comparison between HPLC and UHPLC: mean of results from the Intermediate Precision sets LMM-H Parnaparin Dalteparin Tinzaparin System HPLC UPLC 1 UPLC 2 HPLC UPLC 1 UPLC 2 HPLC UPLC 1 UPLC 2 Mw < 2.0 < > 8.0 (0.29) 27.7 (0.62) 52.8 (0.33) (0.65) 29.1 (1.09) 52.1 (0.56) (0.63) 27.8 (0.97) 52.3 (0.46) (0.29) 10.2 (1.34) 21.1 (0.55) (0.44) 11.5 (3.29) 20.3 (1.55) (0.46) 10.1 (3.15) 21.7 (1.74) (0.67) 6.4 (5.37) 60.4 (0.37) 33.2 (0.65) (1.35) 6.0 (3.33) 61.4 (0.65) 32.5 (1.09) (1.41) 5.5 (3.14) 60.7 (0.57) 33.9 (0.72) 19

20 Ph.Eur LMM-Hs Identification C Transfer results Comparability of results, for most LMM-Hs, is better with Cal Mn = 3900 (UPLC 2 ) The transfer is definitely successful for all Parnap. and Daltep. parameters, while the transfer of Mw only is successful for all other LMM-Hs Discrepancies were found for most MM distribution percentages of Enoxap., Nadrop., Tinzap. The choice of a different Cal Mn, with respect to the official Cal Mn of Ph.Eur., is allowed, in agreement with a Ph.Eur. publication (Pharmeuropa Bio; , 29), supporting the continuity of the measurement system 20

21 USP Enoxaparin Sodium Identification D Operating conditions Column(s) HPLC TSK G3000SW.XL and G2000SW.XL 7.8x300 mm, in series UHPLC WATERS BEH x150 mm 1.7µm Mobile phase 0.5M Lithium Nitrate the same Flow, ml/min Injection Volume, µl Sample / Std conc., mg/ml 10 the same Std USP Enoxaparin Cal A and B RS the same RUN TIME 60 min. 6 min. 21

22 USP Enoxaparin Sodium Identification D Comparison between HPLC and UHPLC: mean of results from the Intermediate Precision sets LMM-H Enoxaparin Sodium RS USP System Suitability (4420 ± 150) Enoxaparin sample (Lovenox) System HPLC UPLC HPLC UPLC Mw 4390 (1.35) 4390 (0.65) 4340 (0.74) 4340 (0.45) Percentage of chains with MM (k) < > (4.08) 71.9 (0.45) 11.1 (4.16) 16.8 (1.84) 72.0 (0.25) 11.2 (1.73) 17.1 (3.42) 72.3 (0.98) 10.6 (2.76) 17.1 (1.96) 72.2 (0.57) 10.8 (1.93) 22

23 USP Enoxaparin Sodium Identification D Transfer results The transfer is definitely successful: comparability of results is excellent and all figures are definitely overlapping 23

24 USP Sodium Heparin Identification D Operating conditions Column(s) HPLC TSK G4000SW.XL and G3000SW.XL 7.8x300 mm, in series UHPLC Waters BEH 450 (2.5µm) and BEH 200 (1.7µm) 2x4.6x150 mm, in series Mobile phase 0.1M Ammonium Acetate the same Flow, ml/min Injection Volume, µl Sample / Std conc., mg/ml 10 the same Std USP Calibrant RS 07/334 the same RUN TIME 60 min. 15 min. 24

25 USP Sodium Heparin Identification D Heparin Heparin Sodium RS USP System Suitability (16000 ± 500) Heparin Sodium Batch 1 Heparin Sodium Batch 2 System HPLC UPLC HPLC UPLC HPLC UPLC Mw (0.25) (0.12) (0.39) (0.26) (0.49) (0.19) Percentage of chains with MM (k) > 24.0 R * 6.9 (2.59) 1.54 (1.13) 7.0 (1.14) 1.53 (0.55) 14.5 (1.70) 1.63 (0.60) 14.7 (0.66) 1.62 (0.18) 10.7 (2.28) 1.99 (1.07) 10.9 (0.73) 1.98 (0.24) * Defined as M / M

26 USP Heparin Sodium Identification D Transfer results The transfer can be considered successful: i) System suitability criteria met ii) All figures are almost equivalent 26

27 USP <209>, draft, for LMM-Hs Operating conditions Column(s) HPLC TSK G3000SW.XL and G2000SW.XL 7.8x300 mm, in series UHPLC Waters BEH x150 mm Mobile phase 0.1M Ammonium Acetate the same Flow, ml/min Injection Volume, µl Sample / Std conc., mg/ml 10 the same Std USP LMW-H Calibrant B the same RUN TIME 60 min. 6 min. 27

28 USP <209>, draft, for LMM-Hs Percentage of chains with MM (k) LMM-H Dalteparin Sodium RS USP System Suitability: 6100 ± 150 Enoxaparin Sodium RS System HPLC UPLC HPLC UPLC Mw 6110 (0.44) < 2.0 < (5.23) > (3.84) 6150 (0.35) 8.6 (6.34) 22.1 (0.98) 4400 (0.64) 14.8 (6.93) 75.2 (1.08) 9.9 (3.82) 4430 (0.31) 13.6 (3.16) 76.3 (0.41) 10.1 (1.29) 28

29 USP <209> draft Transfer results The transfer can be considered successful for the intended purpose (Dalteparin as the only other LMM-H soon in USP): i) System suitability criteria met ii) Figures for Dalteparin almost equivalent Discrepancies were found for most MM distribution percentages of Enoxaparin 29

30 Conclusions WATERS BEH SEC Columns have slightly different sizing performance with respect to TOSOH TSK G SW.XL Differences do not affect the determination of Mw, but, depending on calibration methods and the specific MM distribution, can affect the figures of percentage of chains, especially at the tails of the distribution No clear evidence of differences in the RSD% of the two methods has been shown 30

31 Conclusions Ph. Eur. Identification test C can be successfully transferred to UHPLC for Parnaparin and Dalteparin only. An adjustment of the Cal Mn is required to guarantee the continuity of the measurement system USP Enoxaparin Id. Test D and Heparin Id. Test D can be successfully transferred to UHPLC USP <209> draft, transfer to UHPLC can be considered successful for the intended purpose (Dalteparin), but discrepancies could be found for other LMM-Hs 31

32 Conclusions A successful transfer of all these methods allows a shortening of 4-10 fold of the analysis time This result is of great advantage in all stages of the product development, from the development of new Heparin-related products, to in-process control and Quality Control 32

33 Acknowledgments Opocrin R&D Lab team: Bruschi P., Parma B., Prandi M., Tiddia S., Vismara S. Waters Italy: Cuccurullo M. 33

34 Thank you 34

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