INTRODUCTION:

Low-molecular-weight heparin (LMWH) preparations are widely used to treat and prevent venous thromboembolic complications, including those that may occur in COVID-19 infections.^1-3 LMWHs are widely used to prevent and treat thromboembolic complications associated with veins. They can also be beneficial in complications arising from COVID-19 infection. LMWH differ from conventional heparin in their lower molecular weight. This allows them to bind to antithrombin and selectively block the X-factor of blood clotting. This mechanism of action reduces the risk of bleeding during treatment.^4-6At the same time, the use of LMWHs does not require constant monitoring of coagulation parameters.⁷

They can be used both during pregnancy planning and during pregnancy.^8-10 One such preparation is calcium salt of nadroparin^11,12, which is produced through several stages of chemical processing (Figure 1).

Figure 1: Diagram of the chemical synthesis of calcium nadroparin

During the production of calcium salt of nadroparin, internal control must be carried out at all stages to ensure high quality of the final product. Determining the molecular weight is not difficult, as standard samples manufactured by various manufacturers are available on the market. However, the quantitative determination of nadroparin in the H-form (NadH) at the ion exchange stage is difficult, as there are no standard samples and methods for rapid determination of the H-form in the ion exchange concentrate. In modern practice of assessing the quality of medicinal products, standards are used with which the results of quality control of the drug are compared. This allows determining the compliance of the drug with the established requirements.^13-16

At first glance, it might seem that using the gravimetric method to determine the dry residue of the intermediate product could be effective. However, in conditions of H-form instability and its volatility, the results of gravimetric analysis may be inaccurate, and the lengthy analysis process can lead to degradation of LMWH. Despite the widespread use of reverse-phase chromatography for standardization and quantitative determination of a large number of substances, the option of exclusive chromatography is more applicable for NadH.^17-19 We proposed an alternative method for quantitative determination of the H-form of LMWH at an intermediate stage of ion exchange using HPSEC-RI and the external standard method for calculating the substance content. However, for the external standard method to be used, standard samples are required.^20-24 In this work, we describe the development and certification of CRM NadH. The aim of the work was to create, certify, and determine the stability (shelf life) of CRM NadH for quantitative determination of the H-form of nadroparin by HPSEC-RI as part of improving the control system for the production technology of calcium salt of nadroparin.

MATERIALS AND METHODS:

Materials:

The object of development and certification was the standard sample– H-nadroparin (CRM NadH), obtained on the basis of the Siberian State Medical University. The US Pharmacopoeia reference standard "Low Molecular Weight Heparin Calibrant 10.6 mg" (USP – Low Molecular Weight Heparin Molecular Weight Calibrant RS) cat. No. 1448854, batch No. F0M224 was used as the primary standard.

Production of CRM Nad-H:

The certified standard sample was obtained from calcium nadroparin (APS, C66, produced 2024) by ion exchange using cartridges (250×30 mm) filled with a cation exchange sorbent (AH-50, Biorad), particle size – 125-150 microns, pore size - 200 Å.

For purification, 4.0 ml (25 mg/ml) of calcium nadroparin solution was placed in a pre-cooled cartridge, accumulating eluate. After passing the solution, purified water was added to the syringe, collecting the eluate, until the value of the specific electrical conductivity stabilized. Next, the collected eluate was frozen and freeze-dried (Ilshin freeze-drying, MCFD 8508, South Korea) the temperature of the condenser -80ºС, pressure 0.06 mbar.

Certification of CRM NadH:

a) LMWH content:

The content of the active substance in the NadH samples was determined by the material balance method^25,26 according to the formula:

A = 100 – w_aq– w_sa

where

A –content of low molecular weight heparin, %;

w_aq– water content, %;

w_sa – sulphate ash content, %.

The water content was determined (by the K.Fischer method) using an automatic titrator 915 KF Ti-Touch (Metrohm, USA) and sulfate ash (Pharmacopoeia of the Russian Federation).

b) HPSEC-RI:

The characteristics of the molecular mass distribution were determined using a Dionex Ultimate 3000 liquid chromatograph (Thermo, Germany) with a RI-101 refractometric detector and column TSKgel G2000SW 10 µm 300mm X 7,5mm (Lot№ 0005788).²⁷ Chromatography was performed in isocratic mode, the flow rate was 0.5 ml/min, the column temperature was +30 °C, the volume of the injected sample was 10 µl, the chromatography time was 30 min. A 2.84% solution of sodium sulfate pH =5.0 was used as the mobile phase.

Preparation of the mobile phase. 28.4 g of sodium sulfate was placed in a measuring flask with a capacity of 1000 ml, dissolved in 600 ml of water and the volume was brought to the mark with the same solvent. The pH value was adjusted to 5.0 ± 0.02 using diluted (0.98%) sulfuric acid. They were filtered through a membrane filter with a pore size of 0.45 microns (reduced cellulose).

The solution of the certified standard sample of the enterprise. A sample of 20 mg of the standard was dissolved in 2 ml of the mobile phase, mixed and filtered through a syringe filter 0.45 microns (reduced cellulose).

A solution of a standard sample of low molecular weight heparin (USP). A sample of 10.6 mg of the low molecular weight heparin standard for calibration was dissolved in 1.0 ml of the mobile phase.

Solution for testing the suitability of the chromatographic system. A sample of 100 mg of glycine was placed in a 10 ml volumetric flask, dissolved in the mobile phase, the volume of the flask was brought to the mark with a solvent and mixed.

The chromatographic system was considered suitable if the following conditions were met: on the chromatogram of a solution of a standard sample of low molecular weight heparin, a low molecular weight heparin peak curve was observed, recorded in the range from 12 to 23 minutes; for the main peak on the glycine chromatogram, the column efficiency was at least 6000 theoretical plates; for the main peak on the glycine solution chromatogram, the asymmetry factor was at least 0.8 and no more than 1.5.

The use of low molecular weight substances (e.g. glucose) to test the suitability of the system is due to the complexity of the peak structure of low molecular weight heparins and the inability to determine an acceptable asymmetry factor and the number of theoretical plates. To test the suitability of the chromatographic system, it is proposed to use glycine as a substance that has a high refractive index and moderate absorption in the short-wavelength UV region.

To construct a calibration graph, peak retention times on the chromatogram were used, recorded using a refractometric detector, integrating the low-molecular-weight heparin signal, taking it as a mixture of narrow standards, the molecular weights and contents of which are given in the standard certificate. When integrating the peaks, the solvent and salt signals were not taken into account.

Next, the calibration dependence was constructed in logM_i = f(t_R) coordinates, with a regression dependence of the third order.

On the chromatogram of the solutions of the certified sample, the signal was integrated by dividing it into at least 20 elementary sections, after which the average mass molecular weight (M_w) was calculated:

where:

S_RI,i – the area of the i-th peak on the chromatogram of the test solution;

M_i – the molecular weight of the i-th peak, found from the calibration graph.

Next, the content (X_i, %) of the fraction with a given molecular weight was calculated to compile a specification of the molecular weight distribution of the standard sample.

c) Shelf life study:

The study was carried out under storage conditions of CRM NadH solutions at room temperature, in a refrigerator (2-8 °C)( MPR-414F, Japan), a freezer (-20 °C) (MPR-414F, Japan) and a freezer (-40 °C) (MM-180/20/35 "POZIS", Russia) by comparing chromatograms of the CRM NadH samples with chromatograms of the international standard sample.

RESULT:

a) LMWH content:

Ten series of the standard sample of the enterprise (S.1-S.10) were obtained by ion exchange. The yield of the standard sample ranged from 90 to 95% of the theoretical one. The results of the determination of impurities (water, sulfate ash) and the calculation of the content of NadH is presented in Table 3.

Table 1. Molecular weight and content of controlled fractions of the CRM NadH.

	S.1	S.2	S.3	S.4	S.5	S.6	S.7	S.8	S.9	S.10	Average	RSD (%)
M_w (Da)	4324	4352	4337	4349	4314	4318	4317	4326	4328	4319	4328,3	0,31
Fraction less than 2 kDa (%)	5,48	5,9	5,87	5,51	5,88	5,6	5,65	5,77	5,82	6,09	5,76	3,36
Fraction 2-4 kDa (%)	44,17	43,61	43,49	43,98	43,85	44,47	44,57	43,84	43,74	43,62	43,93	0,83
Fraction 2-8 kDa (%)	86,23	85,45	85,61	86,01	85,94	86,17	85,92	85,85	85,83	85,44	85,85	0,32

Table 2. Molecular weight distribution of the CRM NadH.

Sample	Content (X_i, %) of the fraction of low molecular weight heparin with molecular weight (Mw, Da)
Sample	*18000*	*13600*	*12000*	*9600*	*8400*	*7200*	*6600*	*6000*	*5400*	*4800*	*4200*	*3600*	*3000*	*2400*	*1800*	*1200*
S.1	0,28	0,29	0,33	1,99	2,85	5,02	4,24	4,94	6,72	7,08	10,46	22,27	12,75	13,19	7,15	0,44
S.2	0,38	0,41	0,39	2,04	2,88	4,96	4,23	5,17	6,78	6,76	10,34	21,76	12,74	13,15	7,52	0,49
S.3	0,27	0,31	0,38	2,08	2,93	4,99	4,64	4,9	6,25	7,11	10,63	22,03	12,55	12,95	7,42	0,56
S.4	0,27	0,28	0,36	2,08	2,94	5,13	4,49	5,16	6,72	6,80	10,13	22,53	12,63	12,86	7,14	0,48
S.5	0,30	0,26	0,35	1,87	2,85	5,01	4,12	5,18	6,82	6,80	10,56	22,16	12,90	12,83	7,43	0,56
S.6	0,27	0,25	0,32	1,97	2,87	5,1	4,58	4,35	7,29	6,57	10,21	22,64	12,87	13,00	7,19	0,52
S.7	0,30	0,31	0,34	2,07	2,86	4,96	4,11	5,52	6,09	7,08	9,99	22,12	12,62	13,87	7,19	0,57
S.8	0,29	0,32	0,34	2,04	2,84	5,07	4,85	4,27	6,26	7,49	10,47	21,82	13,00	13,06	7,29	0,59
S.9	0,26	0,31	0,35	2,04	2,84	4,95	5,02	4,1	7,06	6,88	10,48	22,03	12,89	12,86	7,45	0,48
S.10	0,30	0,35	0,36	2,03	2,88	4,92	4,22	4,9	6,38	7,3	10,5	21,49	13,3	12,87	7,61	0,59
Average	0,29	0,31	0,35	2,02	2,87	5,01	4,45	4,85	6,64	6,99	10,38	22,09	12,83	13,06	7,34	0,53
RSD (%)	11,73	14,90	6,11	3,17	1,23	1,38	7,14	9,52	5,79	3,96	1,98	1,57	1,72	2,37	2,30	10,05

Table 3: Results of the certification of the content of low molecular weight heparin in the standard NadH sample

	S.1	S.2	S.3	S.4	S.5	S.6	S.7	S.8	S.9	S.10	Average	RSD (%)
*w_aq (*%)	0,700	0,710	0,720	0,714	0,700	0,698	0,734	0,677	0,689	0,695	0,70	2,32
*w_as (*%)	22,52	22,32	22,59	22,55	22,50	22,32	22,54	22,18	22,47	22,44	22,44	0,58
A (%)	76,78	76,97	76,69	76,74	76,80	76,98	76,73	77,14	76,84	76,87	76,85	0,18

b) HPSEC-RI:

All samples of CRM NadH was analyzed by HPSEC-RI method to assess the molecular weight characteristics according to the calibration dependence constructed according to the international standard sample USP cat. No.1448854 (Figure 2). The correlation coefficient of the trend line equation was more than 0.995.

Figure 2. Calibration dependence of the standard sample of low molecular weight heparin USP RS.

For each batch of the certified standard sample (Fig. 3), the molecular weight (Table 1) and molecular weight characteristics (Table 2) are calculated in 3 parallel definitions.

Figure 3. Chromatogram of sample of CRM Nad-H.

The coefficient of variation within the series ranged from 0.12 to 0.39 %.

The results of the analysis of the molecular weight characteristics of the standard samples presented in Table 2 demonstrate the homogeneity of each sample and the identity of their molecular weight composition. This confirms that CRM Nad-H can be used as an external standard for quantitative determination.

c) Shelf life study:

A comparison of molecular weight characteristics CRM Nad-H during storage for 6 months and USP Calibrant is shown in Figure 4.

Figure 4. Graph of the dependence of the mass average molar mass of the standard Nad-H sample on time.

After investigating the stability of the CRM NadH solution at room temperature, it was decided not to continue this study, since already 2 days after the start of the experiment, the molecular weight value changed by more than 10% relative to the initial one. The study of the stability of solutions during freezing after 180 days was suspended, since we considered the change in molecular weight by more than 5% significant, and the results obtained using these reference samples may be burdened with an error. Apparently, as a result of the desulfurization reaction, the hydrolysis of the polymer molecule occurs, which leads to a decrease in the average molecular weight and an increase in the concentration of free sulfates.

DISCUSSION:

The proposed method for obtaining CRM NadH is convenient and simple, which allows the production of standard samples from both the pharmaceutical substance calcium nadroparin. We obtained 10 samples of CRM and obtained their characteristics.

The values of the relative standard deviation of the molecular weight of the samples and the content of the controlled fractions do not exceed 5.0 % (Table 1), which may indicate the structural similarity of Nad-H and USP heparin calibrant and the suitability of CRM NadH for the control of low molecular weight heparins according to the "molecular weight distribution".

A detailed analysis of the molecular weight distribution (Table 2) shows the "classical" dependence of RSD on the average value with a significant increase up to 15 % for small fraction values (less than 1.0 %). This may be due, first of all, to the adopted regression analysis model (degree 3 polynomial) to cover the entire range of molecular weights of low molecular weight heparins.

The graph of the dependence of the molecular weight shows that the use of "old" solutions is not recommended, which is obvious due to degradation processes. More interesting conclusions can be drawn from the study of the storage process of solutions in a refrigerator and freezer. Thus, for solutions at 2-8 ºС, a significant decrease (more than 10%) in molecular weight was found in less than 3 months, while nadroparin is more stable in the form of salt.¹⁷ No significant difference in molecular weight was determined under experimental conditions at both -20 and -40 ºC, from which it can be concluded that freezer conditions (no higher than -20 ºC) should be accepted as recommended storage conditions for CRM NadH.

The calibration dependence shown in Figure 2 was used to calculate the molecular weight and molecular weight characteristics of all standard CRM samples

CONCLUSION:

Based on the results of the study, a protocol has been developed for obtaining a standard sample of the nadroparin H-form enterprise for in-production control of the sodium nadroparin ion exchange stage in the technology for obtaining calcium nadroparin. The established shelf life of the sample is at least 2 months at temperatures below 8 °C. The stability of solutions of the standard sample at storage temperatures -40 and -20 °C for 6 months is shown.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The work was carried out within the framework of an agreement with the Ministry of Education and Science of Russia dated April 7, 2022 No. 075-11-2022-032

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Received on 07.10.2024 Revised on 10.02.2025

Accepted on 16.04.2025 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2169-2174.

DOI: 10.52711/0974-360X.2025.00311

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