色谱技术在肝素结构分析中的研究进展

doi:10.3724/SP.J.1123.2022.07020

摘要/Abstract

摘要：

肝素(heparin, Hp)是目前临床应用最为广泛的抗凝剂,是由重复二糖单元组成的多硫酸化酸性直链多糖。低分子量肝素(LMWHs)是以肝素为原料,经过化学或酶降解获得的相对分子质量相对较小的肝素衍生物,相对肝素,它们的出血副作用和免疫原性更小,皮下注射时生物利用度更高。肝素及低分子量肝素具有一系列结构特点,如相对分子质量偏大且有一定分布,多种糖残基同时存在,硫酸酯位置和数量呈现多样化,以及不同工艺产生的特殊残基的种类和含量不一等。该类药物结构的复杂性对分析方法提出了巨大的挑战,也限制了其质量控制提升、工艺优化、临床用药安全和新适应证拓展等。该文以色谱分析方法为中心,从结构分析的不同角度,包括单糖、二糖、寡糖、多糖的识别、组成分析和不同层次,系统地梳理和阐述近年来肝素类药物在色谱分析方法上的进展,并对这些方法的应用范畴、创新性、局限性等进行总结。该文将为肝素类药物的结构分析、质量控制提供较系统的方法学参考,为更多新方法开发提供思路,为更深入地研究肝素类药物结构、拓展其应用提供有力支撑。

关键词: 色谱, 单糖组成分析, 二糖组成分析, 寡糖分析, 多糖分析, 肝素, 低分子量肝素, 综述

Abstract:

Heparin (Hp) is the most widely used anticoagulant drug in the clinics, with an annual global output of over 10 billion dollars. Hp, a member of the glycosaminoglycans (GAGs), is prepared from porcine intestinal mucosa via extraction, separation, and purification. Hp is a linear polysaccharide with repeating disaccharide units. Low-molecular-weight heparins (LMWHs) are depolymerized from Hp via chemical or enzymatic degradation. Compared with Hp, LMWHs exhibit less bleeding side effect, milder immunogenicity, and higher bioavailability when injected subcutaneously. In general, Hps, including LMWHs, are high complex drugs with large molecular weights (MWs), inhomogeneous MW distributions, and structural heterogeneity, including different degrees and locations of sulfonation, and unique residues generated from different production processes. Thus, developing efficient analytical methods to elucidate the structures of Hps and characterize or quantitate their properties is extremely challenging. Unfortunately, this problem limits their quality control, production optimization, clinical safety monitoring, and new applications. Research has constantly sought to elucidate the complicated structures of Hp drugs. Among the structural analysis and quality control methods of Hp currently available, chromatographic methods are the most widely studied and used. However, no literature thoroughly summarizes the specific applications of chromatographic methods in the structural analysis, manufacturing process, and quality control of Hp drugs. This paper systematically organizes and describes recent research progresses of the chromatographic methods used to analyze Hp drugs, including the identification and composition of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The applications, innovations, and limitations of these chromatographic methods are also summarized in this review. The insights obtained in this study will help production and quality control personnel, as well as drug researchers, obtain a deeper understanding of the complex structures of Hp drugs. This paper also provides a comprehensive reference for the structural analysis and quality control of Hps, proposes ideas for the development of new quality control methods, and lays a strong foundation for the in-depth structural elucidation of Hp drugs.

Key words: chromatography, monosaccharide composition analysis, disaccharide composition analysis, oligosaccharide analysis, polysaccharide analysis, heparin (Hp), low molecular weight heparin (LMWHs), review

中图分类号:

O658

欧阳艺兰, 易琳, 邱露允, 张真庆. 色谱技术在肝素结构分析中的研究进展[J]. 色谱, 2023, 41(2): 107-121.

OUYANG Yilan, YI Lin, QIU Luyun, ZHANG Zhenqing. Advances in heparin structural analysis by chromatography technologies[J]. Chinese Journal of Chromatography, 2023, 41(2): 107-121.

图/表 10

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No.	Derivatization reagent	Method	Parameters	Analytes	Ref.	Published year
1	PMP	RPLC-UV	LOD:0.04-1.6 μmol/L;LOQ:0.15-1.6 μmol/L;mass recovery:92%-100%;RSD:0.3%	10 sugars	[51]	2016
2	PMP	RPLC-MS(MRM)	LOD:0.056-5.6 fmol/L;LOQ:0.5-10 ng/mL;RSD:6.0%	16 sugars	[53]	2017
3	2-AB and 2-AP	RPLC-UV	LOD:1.2-11 nmol/L;LOQ:4-36 nmol/L;repeatability:2%-9%;inter-day repeatability:3%-9%	10 sugars	[52]	2021
4	d3-4-MOBHA·HCl	RPLC-MS(MRM)	LOD:1.2-11 nmol/L;LOQ:0.25-3 fmol/L;mass recovery: 85%-110%	12 sugars	[58]	2021
5	no	IC	LOD:1.0 ng;LOQ:2.5 ng	16 sugars	[45]	2012

No.	Derivatization reagent	Method	Parameters	Analytes	Ref.	Published year
1	PMP	RPLC-UV	LOD:0.04-1.6 μmol/L;LOQ:0.15-1.6 μmol/L;mass recovery:92%-100%;RSD:0.3%	10 sugars	[51]	2016
2	PMP	RPLC-MS(MRM)	LOD:0.056-5.6 fmol/L;LOQ:0.5-10 ng/mL;RSD:6.0%	16 sugars	[53]	2017
3	2-AB and 2-AP	RPLC-UV	LOD:1.2-11 nmol/L;LOQ:4-36 nmol/L;repeatability:2%-9%;inter-day repeatability:3%-9%	10 sugars	[52]	2021
4	d3-4-MOBHA·HCl	RPLC-MS(MRM)	LOD:1.2-11 nmol/L;LOQ:0.25-3 fmol/L;mass recovery: 85%-110%	12 sugars	[58]	2021
5	no	IC	LOD:1.0 ng;LOQ:2.5 ng	16 sugars	[45]	2012

No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]	IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]		HILIC- MS^Eno[60]
1	ΔU-GlcNAc	1	1	1	1	1		1		1
2	ΔU-GlcNS	1	1	1	1	1		1		1
3	ΔU-GlcNAc,6S	1	1	1	1	1		1		1
4	ΔU2S-GlcNAc	1	1	1	1	1		1		1
5	ΔU-GlcNS,6S	1	1	1	1	1		1		1
6	ΔU2S-GlcNS	1	1	1	1	1		1		1
7	ΔU2S-GlcNAc,6S	1	1	1	1	1		1		1
8	ΔU2S-GlcNS,6S	1	1	1	1	1		1		1
9	ΔGalA-GlcNS	1	0	0	0	0		1		1
10	ΔGalA-GlcNS,6S	1	0	0	0	0		1		1
12	IdoA2S-GlcNS	1	0	0	0	0		1		1
13	U2S-GlcNS or U-GlcNS,6S	0	0	1	0	0		0		0
14	U2S-GlcNAc or U-GlcNAc,6S	0	0	0	1	0		0		0
15	IdoA2S-GlcNS,6S	1	0	0	0	0		1		1
16	ΔU2S-GlcN,6S	0	0	0	0	0		1		1
17	ΔU2S-GlcN	0	0	0	0	0		1		1
18	ΔU-GlcN,6S	0	0	0	0	0		1		1
19	ΔU-GlcN	0	0	0	0	0		0		1
20	ΔGlyser	1	1	1	1	0		1		1
No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]		IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]	HILIC- MS^Eno[60]
21	ΔGlyser_ox1	1	0	0	0		0		1	1
22	ΔGlyser_ox2	1	0	0	0		0		0	0
23	Other non-endogenous derivatives ΔI_SO3	1	0	0	0		0		0	0
24	Tetrasaccharide A ΔI_S-I_2SO3	1	0	0	0		0		0	0
25	Tetrasaccharide B ΔI_2SO3-I_sid	1	0	0	0		0		0	0
26	ΔU-GlcNAc,6S-GlcA-GlcNS,3S	1	0	0	0		0		0	0
27	ΔU-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	1	1	1		1		1	1
28	ΔU-GlcNS,6S-GlcA-GlcNS,3S,6S	1	1	1	0		0		1	1
29	ΔU2S-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
30	ΔU2S-GlcNS,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
31	GlcNS-IdoA2S-GlcNS,6S or GlcNS,	0	0	1	0		0		0	0
	6S-IdoA2S-GlcNS
32	GlcNS,6S-IdoA2S-GlcNS,6S	1	0	0	0		0		1	1
33	ΔU-GlcNS,6S-HexA	0	0	0	0		0		1	1
34	ΔU2S-GlcNAc,6S-HexA	0	0	0	0		0		0	1
35	ΔU2S-GlcNS,6S-HexA	0	0	0	0		0		1	1
36	ΔU2S-GlcNS,6S-HexA2S	0	0	1	0		0		1	1
37	ΔU2CS-GlcNS,6S	0	0	0	0		0		1	1
38	GlcNS,6S-U2CS-GlcNS or GlcNS-U2CS-GlcNS,6S	0	0	0	1		0		0	0
39	ΔUA2S-GlcNS,6S-GlcA-2,3-anhydro-GlcNS	0	0	0	0		0		1	1
40	ΔUA-GlcNS-HexA2S,3S-GlcNS	0	0	0	0		0		1	1
41	3-O-S Δdp2	0	0	0	0		0		1	0
42	3-O-S Δdp4(3S,1Ac)	0	0	0	0		0		1	0
43	3-O-S Δdp4(4S,0Ac)	0	1	1	0		0		1	0
44	3-O-S Δdp4(4S,1Ac)	0	2	0	0		0		0	0
45	3-O-S Δdp4(5S,0Ac)	0	3	0	0		0		0	0
46	ΔU- Mnt6S	0	0	0	1		0		1	0
47	ΔU2S- Mnt6S	0	0	0	1		0		1	0
48	GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		0	0
49	ΔU2S-GlcNS-U-Mnt6S	0	0	0	0		0		1	0
50	ΔU2S-GlcNAc-U2S-Mnt6S	0	0	0	1		0		0	0
51	ΔU2S-GlcNS-U2S-Mnt6S	0	0	0	1		0		1	0
52	ΔU2S-GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		1	0
53	MntΔdp5(5S,1Ac)	0	0	0	1		0		0	0
54	MntΔdp6(6S,1Ac)	0	0	0	1		0		0	0
55	ΔU-1,6-anhydroGlcNS	0	0	0	0		0		0	1
56	ΔU-1,6-anhydroManNS	0	0	0	0		0		0	1
57	ΔU2S-1,6-anhydroHexNS	0	0	0	0		2		0	1
58	GlcNS,6S-U2S-1,6-anhydroHexNS	0	0	1	0		0		0	0
59	ΔU2S-GlcNS,6S-U2S-1,6-anhydroManNS	0	0	0	0		1		0	1

No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]	IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]		HILIC- MS^Eno[60]
1	ΔU-GlcNAc	1	1	1	1	1		1		1
2	ΔU-GlcNS	1	1	1	1	1		1		1
3	ΔU-GlcNAc,6S	1	1	1	1	1		1		1
4	ΔU2S-GlcNAc	1	1	1	1	1		1		1
5	ΔU-GlcNS,6S	1	1	1	1	1		1		1
6	ΔU2S-GlcNS	1	1	1	1	1		1		1
7	ΔU2S-GlcNAc,6S	1	1	1	1	1		1		1
8	ΔU2S-GlcNS,6S	1	1	1	1	1		1		1
9	ΔGalA-GlcNS	1	0	0	0	0		1		1
10	ΔGalA-GlcNS,6S	1	0	0	0	0		1		1
12	IdoA2S-GlcNS	1	0	0	0	0		1		1
13	U2S-GlcNS or U-GlcNS,6S	0	0	1	0	0		0		0
14	U2S-GlcNAc or U-GlcNAc,6S	0	0	0	1	0		0		0
15	IdoA2S-GlcNS,6S	1	0	0	0	0		1		1
16	ΔU2S-GlcN,6S	0	0	0	0	0		1		1
17	ΔU2S-GlcN	0	0	0	0	0		1		1
18	ΔU-GlcN,6S	0	0	0	0	0		1		1
19	ΔU-GlcN	0	0	0	0	0		0		1
20	ΔGlyser	1	1	1	1	0		1		1
No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]		IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]	HILIC- MS^Eno[60]
21	ΔGlyser_ox1	1	0	0	0		0		1	1
22	ΔGlyser_ox2	1	0	0	0		0		0	0
23	Other non-endogenous derivatives ΔI_SO3	1	0	0	0		0		0	0
24	Tetrasaccharide A ΔI_S-I_2SO3	1	0	0	0		0		0	0
25	Tetrasaccharide B ΔI_2SO3-I_sid	1	0	0	0		0		0	0
26	ΔU-GlcNAc,6S-GlcA-GlcNS,3S	1	0	0	0		0		0	0
27	ΔU-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	1	1	1		1		1	1
28	ΔU-GlcNS,6S-GlcA-GlcNS,3S,6S	1	1	1	0		0		1	1
29	ΔU2S-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
30	ΔU2S-GlcNS,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
31	GlcNS-IdoA2S-GlcNS,6S or GlcNS,	0	0	1	0		0		0	0
	6S-IdoA2S-GlcNS
32	GlcNS,6S-IdoA2S-GlcNS,6S	1	0	0	0		0		1	1
33	ΔU-GlcNS,6S-HexA	0	0	0	0		0		1	1
34	ΔU2S-GlcNAc,6S-HexA	0	0	0	0		0		0	1
35	ΔU2S-GlcNS,6S-HexA	0	0	0	0		0		1	1
36	ΔU2S-GlcNS,6S-HexA2S	0	0	1	0		0		1	1
37	ΔU2CS-GlcNS,6S	0	0	0	0		0		1	1
38	GlcNS,6S-U2CS-GlcNS or GlcNS-U2CS-GlcNS,6S	0	0	0	1		0		0	0
39	ΔUA2S-GlcNS,6S-GlcA-2,3-anhydro-GlcNS	0	0	0	0		0		1	1
40	ΔUA-GlcNS-HexA2S,3S-GlcNS	0	0	0	0		0		1	1
41	3-O-S Δdp2	0	0	0	0		0		1	0
42	3-O-S Δdp4(3S,1Ac)	0	0	0	0		0		1	0
43	3-O-S Δdp4(4S,0Ac)	0	1	1	0		0		1	0
44	3-O-S Δdp4(4S,1Ac)	0	2	0	0		0		0	0
45	3-O-S Δdp4(5S,0Ac)	0	3	0	0		0		0	0
46	ΔU- Mnt6S	0	0	0	1		0		1	0
47	ΔU2S- Mnt6S	0	0	0	1		0		1	0
48	GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		0	0
49	ΔU2S-GlcNS-U-Mnt6S	0	0	0	0		0		1	0
50	ΔU2S-GlcNAc-U2S-Mnt6S	0	0	0	1		0		0	0
51	ΔU2S-GlcNS-U2S-Mnt6S	0	0	0	1		0		1	0
52	ΔU2S-GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		1	0
53	MntΔdp5(5S,1Ac)	0	0	0	1		0		0	0
54	MntΔdp6(6S,1Ac)	0	0	0	1		0		0	0
55	ΔU-1,6-anhydroGlcNS	0	0	0	0		0		0	1
56	ΔU-1,6-anhydroManNS	0	0	0	0		0		0	1
57	ΔU2S-1,6-anhydroHexNS	0	0	0	0		2		0	1
58	GlcNS,6S-U2S-1,6-anhydroHexNS	0	0	1	0		0		0	0
59	ΔU2S-GlcNS,6S-U2S-1,6-anhydroManNS	0	0	0	0		1		0	1