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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
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International Symposium on GPC/SEC and Related Techniques October, 1st 2014 - Frankfurt
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
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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
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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
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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
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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
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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
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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
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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, LiNO3 or Na2SO4 solutions, 0.1 - 0.5 M
• Flow rates 0.5 or 0.6 mL/min • Run times from 30 min (single column) to 60 min
(coupled columns)
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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
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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
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Ph.Eur. general monograph 0828 for LMM-Hs Identification “C”
• Column: single, silica based, fractionation range 15-100 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
Eur.Ph. specifications from the specific monographs of the 5 LMM-Hs, Identification “C”
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LMM-Heparin Enoxaparin Nadroparin Parnaparin Dalteparin Tinzaparin
Mw ( kDa ) 3.8 – 5.0 3.6 – 5.0 4.0 – 6.0 5.6 – 6.4 5.5 – 7.5
Perc
enta
ge o
f cha
ins
with
MM
(kD
a)
< 2.0 12 – 20 NMT 15 - - NMT 10
< 3.0 - - NMT 30 NMT 13 -
2.0 - 4.0 - 35 – 55 - - -
2.0 - 8.0 68 – 82 75 – 95 - - 60 – 72
3.0 - 8.0 - - 50 – 60 - -
> 8.0 - - - 15 – 25 22 – 36
Ph.Eur. 0828 - LMM-H Identification “C”
Operating conditions
HPLC UHPLC
Column(s) TSK G2000SW.XL (5µm) 7.8x300 mm
WATERS BEH 200 4.6x150 mm 1.7µm
Mobile phase 0.2M sodium sulphate, pH 5.0 the same
Flow, mL/min 0.5 0.4
Injection Volume, µL 25 2.5
Sample / Std conc., mg/mL 10 the same
Std LMM-CRS Batch 3 the same
RUN TIME 30 min. 6 min.
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Method transfer: Ph.Eur. 0828 – LMM-Hs Identification “C”
5016M
V
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00
LMW
_H -
3.89
0
µRIU
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
LMM-H CRS, HPLC
LMM-H CRS, UHPLC
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Method transfer: Ph.Eur. 0828 – 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
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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)
4310 (0.71)
4420 (0.63)
4680 (0.29)
4550 (0.48)
4670 (0.51)
5480 (0.29)
5350 (0.65)
5490 (0.63)
Perc
enta
ge o
f cha
ins w
ith
MM
(k)
< 2.0 17.6 (1.95)
16.9 (2.37)
15.7 (2.51)
7.1 (6.02)
6.6 (5.37)
5.9 (4.97)
< 3.0 27.7 (0.62)
29.1 (1.09)
27.8 (0.97)
2.0 - 4.0 41.7 (0.74)
45.6 (2.16)
44.2 (2.02)
2.0 - 8.0 72.2 (0.37)
73.2 (0.86)
73.7 (0.83)
83.0 (0.46)
84.0 (0.51)
84.0 (0.51)
3.0 - 8.0 52.8
(0.33) 52.1 (0.56)
52.3 (0.46)
Comparison between HPLC and UHPLC: mean of results from the Intermediate Precision sets
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Method transfer: Ph.Eur. 0828 – LMM-Hs Identification “C”
Comparison between HPLC and UHPLC: mean of results from the Intermediate Precision sets
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Method transfer: Ph.Eur. 0828 – LMM-Hs Identification “C”
LMM-H Parnaparin Dalteparin Tinzaparin System HPLC UPLC 1 UPLC 2 HPLC UPLC 1 UPLC 2 HPLC UPLC 1 UPLC 2
Mw 5480 (0.29)
5350 (0.65)
5490 (0.63)
6090 (0.29)
5940 (0.44)
6100 (0.46)
7240 (0.67)
7060 (1.35)
7230 (1.41)
Perc
enta
ge o
f cha
ins w
ith
MM
(k)
< 2.0 6.4 (5.37)
6.0 (3.33)
5.5 (3.14)
< 3.0 27.7 (0.62)
29.1 (1.09)
27.8 (0.97)
10.2 (1.34)
11.5 (3.29)
10.1 (3.15)
2.0 - 8.0 60.4 (0.37)
61.4 (0.65)
60.7 (0.57)
3.0 - 8.0 52.8 (0.33)
52.1 (0.56)
52.3 (0.46)
> 8.0 21.1
(0.55) 20.3 (1.55)
21.7 (1.74)
33.2 (0.65)
32.5 (1.09)
33.9 (0.72)
Ph.Eur. 0828 – 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; 2007-1, 29), supporting the continuity of the measurement system
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USP Enoxaparin Sodium Identification “D”
Operating conditions HPLC UHPLC
Column(s) TSK G3000SW.XL and G2000SW.XL 7.8x300 mm, in series
WATERS BEH 200 4.6x150 mm 1.7µm
Mobile phase 0.5M Lithium Nitrate the same
Flow, mL/min 0.6 0.4
Injection Volume, µL 25 2.5
Sample / Std conc., mg/mL 10 the same
Std USP Enoxaparin Cal A and B RS the same
RUN TIME 60 min. 6 min.
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USP Enoxaparin Sodium Identification “D” Comparison between HPLC and UHPLC: mean of results from the Intermediate Precision sets
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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)
Perc
enta
ge o
f ch
ains
with
MM
(k
)
< 2.0 17.0 (4.08)
16.8
(1.84) 17.1 (3.42)
17.1 (1.96)
2.0 - 8.0 71.9 (0.45)
72.0 (0.25)
72.3 (0.98)
72.2 (0.57)
> 8.0 11.1 (4.16)
11.2 (1.73)
10.6
(2.76) 10.8 (1.93)
USP Enoxaparin Sodium Identification “D” Transfer results
• The transfer is definitely successful: comparability of results is excellent and all figures are definitely overlapping
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USP Sodium Heparin Identification “D”
Operating conditions HPLC UHPLC
Column(s) TSK G4000SW.XL and G3000SW.XL 7.8x300 mm, in series
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 0.6 0.3
Injection Volume, µL 25 2.5
Sample / Std conc., mg/mL 10 the same
Std USP Calibrant RS 07/334 the same
RUN TIME 60 min. 15 min.
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USP Sodium Heparin Identification “D”
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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 15570 (0.25)
15630 (0.12)
16520 (0.39)
16610 (0.26)
14900 (0.49)
14970 (0.19)
Perc
enta
ge o
f cha
ins
with
MM
(k)
> 24.0 6.9
(2.59) 7.0
(1.14) 14.5 (1.70)
14.7 (0.66)
10.7 (2.28)
10.9 (0.73)
R * 1.54 (1.13)
1.53 (0.55)
1.63 (0.60)
1.62 (0.18)
1.99 (1.07)
1.98 (0.24)
* Defined as M8000-16000 / M16000-24000
USP Heparin Sodium Identification “D” Transfer results
• The transfer can be considered successful: i) System suitability criteria met ii) All figures are almost equivalent
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USP <209>, draft, for LMM-Hs
Operating conditions HPLC UHPLC
Column(s) TSK G3000SW.XL and G2000SW.XL 7.8x300 mm, in series
Waters BEH 200 4.6x150 mm
Mobile phase 0.1M Ammonium Acetate the same
Flow, mL/min 0.5 0.4
Injection Volume, µL 25 2.5
Sample / Std conc., mg/mL 10 the same
Std USP LMW-H Calibrant B130159 the same
RUN TIME 60 min. 6 min.
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USP <209>, draft, for LMM-Hs
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LMM-H Dalteparin Sodium RS
USP System Suitability: 6100 ± 150
Enoxaparin Sodium RS
System HPLC UPLC HPLC UPLC
Mw 6110 (0.44)
6150 (0.35)
4400 (0.64)
4430 (0.31)
Perc
enta
ge o
f cha
ins
with
MM
(k)
< 2.0 14.8 (6.93)
13.6 (3.16)
< 3.0 9.1 (5.23)
8.6 (6.34)
2.0-8.0 75.2 (1.08)
76.3 (0.41)
> 8.0 21.6 (3.84)
22.1 (0.98)
9.9 (3.82)
10.1 (1.29)
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
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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
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• 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
Conclusions
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• 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
Conclusions
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Acknowledgments
• Opocrin R&D Lab team: Bruschi P., Parma B., Prandi M., Tiddia S., Vismara S.
• Waters Italy: Cuccurullo M.
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Thank you
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