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Critical Quality Control Methods for a Novel Anticoagulant Candidate LFG-Na by HPSEC-MALLS-RID and Bioactivity Assays.
Zheng S
,
Wang Y
,
Wu J
,
Wang S
,
Wei H
,
Zhang Y
,
Zhou J
,
Shi Y
.
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A low molecular weight fucosylated glycosaminoglycan sodium (LFG-Na) is a novel anticoagulant candidate from the sea cucumber Holothuria fuscopunctata that selectively inhibits intrinsic tenase (iXase). The molecular weight, molecular weight distribution and bioactivities are the critical quality attributes of LFG-Na. The determination of these quality attributes of such an oligosaccharides mixture drug is challenging but critical for the quality control process to ensure its safety and efficacy in clinical use. Herein, the molecular weight and molecular weight distribution of LFG-Na were successfully determined using high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector (HPSEC-MALLS-RID). Comparing to the conventional method, HPSEC-MALLS-RID based on the refractive index increment (dn/dc) did not require the reference substances to establish the calibration curve. The acceptance criteria of LFG-Na were established, the weight-average molecular weight (Mw) should be 4000 to 6000 Da, the polydispersity (Mw/Mn) < 1.40, and the fraction with molecular weights of 1500 to 8000 Da should be no less than 80% of the total. HPSEC-MALLS-RID was also utilized for the determination of the starting material native fucosylated glycosaminoglycan (NFG) to choose a better manufacturing process. Furthermore, APTT assay was selected and the potency of anti-iXase, referring to the parallel line assay (PLA) method, was established to clarify the consistency of its biological activities. The results suggest that HPSEC-MALLS-RID and bioactivity assays are critical quality control methods for multi-component glycosaminoglycan LFG-Na. The methods also provide a feasible strategy to control the quality of other polysaccharide medicines.
2018ZX09711-001 the National Major Scientific and Technological Special Project for Significant New Drugs Development, ZC2018SH0004 Transformation Project of Major Scientific and Technological Achievements of Heilongjiang Province, 2020SK2043 Special Funding for the Construction of Innovative Provinces in Hunan Province, HHCF20180101 Industry Incubation and Cluster Innovation Project of Marine Economic Innovation and Development Demonstration City of Haikou
Figure 1. Chromatogram obtained from the determination of dn/dc value (LFG-Na, batch L3, concentrations of LFG-Na solutions-differential refractive index were fitted, the Fit R2 = 1.0000 from ASTRA software (version 7.1.3, Wyatt Technology Co., Santa Barbara, CA, USA)).
Figure 2. Representative HPSEC chromatogram of NFG (A) and NFG-2.0M (B) obtained before the NFG manufacturing process optimization from the determination of HPSEC-MALLS-RID.
Figure 3. Representative HPSEC-MALLS-RID chromatogram of LFG-Na.
Figure 4. HPGPC profiles of LFG-Na and oligosaccharide standards HS5, HS8, HS11, HS14, HS17, which were determined by the differential refractive index detector (A). The distribution plot of LFG-Na was analyzed by using Agilent GPC/SEC software (version A.02.01, Agilent Technologies, Palo Alto, CA, USA) (B).
Figure 5. The results of molecular weight (A) and molecular weight distribution (B) for LFG-Na by HPGPC and HPSEC-MALLS-RID.
Figure 6. HPLC profiles of PMP derivatives from LFG-Na (L1–L5).
Figure 7. The structure of LFG-Na (A), 1D 1H NMR spectra (B) and 13C NMR spectra (C) from five batches of LFG-Na (L1–L5). The letters of (A) between parentheses are used as labels for assignments in the 1H-NMR (B) and 13C-NMR spectrum (C). They are the following: dU for the nonreducing terminal unsaturated uronic acid residue; U for internal glucuronic acid; rU for reducing terminal alcohol; dA for GalNAc4S6S linked to dU; A for internal GalNAc4S6S; rA for GalNAc4S6S linked to rU; dF for Fuc3S4S residue linked to dU; F for Fuc3S4S residue linked to U; rF for Fuc3S4S residue linked to rU. 1D 1H (B) and 13C (C) NMR spectra are from five batches of LFG-Na (L1–L5).
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