Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення
The aim of the study was to adjust and optimize the purification stage of crude enoxaparin sodium to obtain a substance equivalent to the original drugs Clexane® and Lovenox® according to the criteria specified by the FDA. The purification stage involves the reprecipitation of crude enoxaparin in me...
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| Published in: | Журнал органічної та фармацевтичної хімії |
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| Date: | 2023 |
| Volume: | 21 |
| Issue: | 3 |
| Pages: | 38-49 |
| ISSN: | 2518-1548 |
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| Main Authors: | , , , , |
| Format: | Article |
| Language: | English |
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| Online Access: | https://ophcj.nuph.edu.ua/article/view/290670 |
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Journal of Organic and Pharmaceutical Chemistry| _version_ | 1874364463182774272 |
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| author | Bovsunovska, Yuliia V. Rudiuk, Vitalii V. Harna, Nataliia V. Holovchenko, Olha S. Georgiyants, Victoriya A. |
| author_facet | Bovsunovska, Yuliia V. Rudiuk, Vitalii V. Harna, Nataliia V. Holovchenko, Olha S. Georgiyants, Victoriya A. |
| author_institution_txt_mv | [
{
"author": "Yuliia V. Bovsunovska",
"institution": "JSC Farmak",
"orcid": ""
},
{
"author": "Vitalii V. Rudiuk",
"institution": "JSC Farmak",
"orcid": "0000-0003-3440-1139"
},
{
"author": "Nataliia V. Harna",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
},
{
"author": "Olha S. Holovchenko",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
},
{
"author": "Victoriya A. Georgiyants",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
}
] |
| author_orcid_str_mv | 0000-0003-3440-1139 |
| author_sort | Bovsunovska, Yuliia V. |
| baseUrl_str | https://ophcj.nuph.edu.ua/oai |
| collection | OJS |
| container_end_page | 49 |
| container_issue | 3 |
| container_start_page | 38 |
| container_title | Журнал органічної та фармацевтичної хімії |
| container_volume | 21 |
| datestamp_date | 2026-08-23T18:59:42Z |
| description | The aim of the study was to adjust and optimize the purification stage of crude enoxaparin sodium to obtain a substance equivalent to the original drugs Clexane® and Lovenox® according to the criteria specified by the FDA. The purification stage involves the reprecipitation of crude enoxaparin in methanol. Determining the ratio of solvents required for the reprecipitation is important for studying the correlation between the experimental conditions of the technological process and the structural characteristics of enoxaparin samples. In the study, the method of purification of enoxaparin sodium described in the patent was assessed, and the following variations of the MeOH:H2O solvent ratio were selected – 4:1; 2:1; 1:1. The obtained samples of enoxaparin sodium were analyzed according to the in-house specification developed on the basis of the pharmacopoeial monograph, as well as by non-pharmacopoeial methods, such as two-dimensional NMR spectroscopy (HSQC) and size exclusion chromatography (SEC) for detailed characterization of the molecule. Strategies of greening of the enoxaparin sodium purification stage by reducing the E-factor were also considered in the study. Considering the principles of “green” chemistry, the method of purification of crude enoxaparin sodium was optimized by the solvent regeneration. It was experimentally possible to demonstrate the effect of the solvent ratio at the stage of purification of crude enoxaparin on the composition, as well as on the number and distribution of oligosaccharide fractions in the molecule. Based on the results of the study, it can be concluded that the ratio of MeOH:H2O=1:1 allows obtaining samples that are closest to Clexane® and Lovenox® in terms of the molecular weight distribution profile and the composition profile. The E-factor was also reduced from 14 to 5.25 by solvent regeneration. |
| doi_str_mv | 10.24959/ophcj.23.290670 |
| first_indexed | 2025-07-23T04:43:23Z |
| format | Article |
| fulltext |
ISSN 2308-8303 (Print) / 2518-1548 (Online) 38
Original Research
http://ophcj.nuph.edu.ua
UDC 661.1:615.4
Y. V. Bovsunovska1,2, V. V. Rudiuk2, N. V. Harna1, O. S. Holovchenko1, V. A. Georgiyants1
1 National University of Pharmacy of the Ministry of Health of Ukraine,
53 Pushkinska str., 61002 Kharkiv, Ukraine
2 JSC Farmak, 63 Kyrylivska str., 04080 Kyiv, Ukraine
Obtaining the Enoxaparin Sodium Substance Equivalent to the
Original Clexane® and Lovenox®. The Selection of Technological
Parameters and Optimization of the “Greenness” of the
Purification Stage
Abstract
The aim of the study was to adjust and optimize the purification stage of crude enoxaparin sodium to obtain a substance
equivalent to the original drugs Clexane® and Lovenox® according to the criteria specified by the FDA. The purification stage
involves the reprecipitation of crude enoxaparin in methanol. Determining the ratio of solvents required for the reprecipi-
tation is important for studying the correlation between the experimental conditions of the technological process and the
structural characteristics of enoxaparin samples. In the study, the method of purification of enoxaparin sodium described in
the patent was assessed, and the following variations of the MeOH:H2O solvent ratio were selected – 4:1; 2:1; 1:1. The ob-
tained samples of enoxaparin sodium were analyzed according to the in-house specification developed on the basis of
the pharmacopoeial monograph, as well as by non-pharmacopoeial methods, such as two-dimensional NMR spectroscopy
(HSQC) and size exclusion chromatography (SEC) for detailed characterization of the molecule. Strategies of greening of the
enoxaparin sodium purification stage by reducing the E-factor were also considered in the study. Considering the principles
of “green” chemistry, the method of purification of crude enoxaparin sodium was optimized by the solvent regeneration. It
was experimentally possible to demonstrate the effect of the solvent ratio at the stage of purification of crude enoxaparin on
the composition, as well as on the number and distribution of oligosaccharide fractions in the molecule. Based on the results
of the study, it can be concluded that the ratio of MeOH:H2O=1:1 allows obtaining samples that are closest to Clexane® and
Lovenox® in terms of the molecular weight distribution profile and the composition profile. The E-factor was also reduced
from 14 to 5.25 by solvent regeneration.
Keywords: enoxaparin sodium; low-molecular-weight heparin; technological parameters; compositional analysis; HSQC;
size-exclusion chromatography; green chemistry; E-factor; solvent regeneration
Ю. В. Бовсуновська1,2, В. В. Рудюк2, Н. В. Гарна1, О. С. Головченко1, В. А. Георгіянц1
1 Національний фармацевтичний університет Міністерства охорони здоров’я України,
вул. Пушкінська, 53, м. Харків, 61002, Україна
2 АТ «Фармак», вул. Кирилівська, 63, м. Київ, 04080, Україна
Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®.
Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення
Анотація
Метою роботи було налаштувати та оптимізувати стадію очищення технічного еноксапарину натрію для отримання
субстанції, еквівалентної оригінальним препаратам Clexane® та Lovenox® за критеріями, окресленими FDA. Стадія
очищення передбачає переосадження неочищеного еноксапарину із метанолу. Визначення необхідного співвідно-
шення розчинників для переосадження є важливим для дослідження кореляції між експериментальними умовами
технологічного процесу та структурними характеристиками зразків еноксапарину. У дослідженні було оцінено спосіб
очищення еноксапарину натрію, описаний у патенті, і обрано такі варіанти співвідношення розчинників MeOH:H2O
– 4:1; 2:1; 1:1. Отримані зразки еноксапарину натрію аналізували відповідно до внутрішньої специфікації, розробле-
ної на основі фармакопейної монографії, а також за допомогою нефармакопейних методів, таких, як двовимірна
ЯМР-спектроскопія (HSQC) та ексклюзійна хроматографія (SEC) для детальної характеристики. молекули. У дослідженні
також розглядали стратегії екологізації етапу очищення еноксапарину натрію шляхом зниження Е-фактора. З огляду
ISSN 2308-8303 (Print) / 2518-1548 (Online) 39
Journal of Organic and Pharmaceutical Chemistry 2023, 21 (3)
на принципи «зеленої» хімії метод очищення неочищеного еноксапарину натрію було оптимізовано шляхом реге-
нерації розчинника. Експериментально вдалося продемонструвати вплив співвідношення розчинників на стадії очи-
щення неочищеного еноксапарину на склад, а також на кількість і розподіл фракцій олігосахаридів у молекулі. За ре-
зультатами дослідження можна зробити висновок, що співвідношення MeOH:H2O = 1:1 дозволяє отримати зразки,
які за профілем молекулярно-масового розподілу та профілем складу найбільш наближені до Clexane® та Lovenox®.
Е-коефіцієнт також було знижено з 14 до 5,25 шляхом регенерації розчинника.
Ключові слова: еноксапарин натрію; низькомолекулярний гепарин; технологічні параметри; композиційний аналіз;
HSQC; ексклюзійна хроматографія; «зелена» хімія; Е-фактор; регенерація розчинника
Citation: Bovsunovska, Y. V.; Rudiuk, V. V.; Harna, N. V.; Holovchenko, O. S.; Georgiyants, V. A. Obtaining the Enoxaparin Sodium
Substance Equivalent to the Original Clexane® and Lovenox®. The Selection of Technological Parameters and Optimization of the
“Greenness” of the Purification Stage. Journal of Organic and Pharmaceutical Chemistry 2023, 21 (3), 38 – 49.
https://doi.org/10.24959/ophcj.23.290670
Received: 3 October 2023; Revised: 29 October 2023; Accepted: 4 November 2023
Copyright© 2023, Yu. V. Bovsunovska, V. V. Rudiuk, N. V. Harna, O. S. Holovchenko, V. A. Georgiyants. This is an open access article under
the CC BY license (http://creativecommons.org/licenses/by/4.0).
Funding: The research was carried out with the financial support of JSC Farmak (Kyiv, Ukraine).
Conflict of interests: The authors declare that they have no conflict of interest in relation to this study, including financial, personal,
authorship, or any other, that could affect the study and its results presented in this article.
■ Introduction
Enoxaparin sodium is a modern low-molecu
lar semi-synthetic anticoagulant, which is a pro-
duct of the multi-stage conversion of sodium he-
parin [1]. Enoxaparin sodium has the same pro-
perties as its precursor heparin sodium, but due
to its improved structure, it does not have the side
effects typical of heparin caused by its complex
structure and very high molecular weight [2, 3].
Enoxaparin sodium is a heterogeneous mix-
ture of oligosaccharides with a complex structu-
re consisting of repeating units of disaccharide
building blocks with one glucuronic acid (GlcA)
or iduronic acid (IdoA) residue and one glucosa-
mine (GlcN) residue, which is either N-sulfated
(GlcNS), or N-acetylated (GlcNAc), linked by gly-
cosidic bonds. Enoxaparin sodium is characteri-
zed by unique structural elements (fingerprints)
that are formed because of modifications during
depolymerization, namely, such structures as
4,5-uronates at non-reducing ends and 1,6-an-
hydro structures at reducing ends [4, 5]. Enoxa-
parin sodium is a substance of biological origin,
i.e., isolated from animal tissues and differs from
“normal” substances by its high molecular weight
and complex heterogeneous structure, which com-
plicates the development and introduction of si-
milar drugs to the market. Due to the complex-
ity of the structure and the previous experience
during the heparin crisis [6], there are serious
discussions in the world on the issues of proving
the equivalence of generic low-molecular-weight
heparins (LMWHs) and establishing permissible
fluctuations of the “norm” of biochemical and bi-
ological indicators, which potentially affect the
safety and effectiveness of the drug. As a result,
EMA and FDA have initiated guidelines to con-
firm the similarity of LMWH [7, 8]. The FDA,
for example, introduced a scientific approach to
demonstrate the equivalence of generic LMWHs
to references, which included compliance not only
with biological, but also with chemical characte-
ristics, such as the sequence of disaccharide buil-
ding blocks, the sequence of oligosaccharide frag-
ments, etc. [9]. Since the aim of our work was
the synthesis of the Enoxparin molecule demon-
strating the equivalence to the original Clexane®
and Lovenox® (Sanofi-Aventis) according to the
specified FDA criteria [10], we conducted a large
study to adjust the technological parameters of
the process at each stage to obtain a substance as
close as possible to the originator [11]. The me-
thods described in the patent [12] were taken as
a basis. The analysis of samples for comparison
was carried out according to the internal speci-
fication developed based on the pharmacopoeial
monograph, as well as according to specific me-
thods. Since the structure requires accurate, pains-
taking analysis of saccharide units and their se-
quence, additional methods of analysis of simi-
lar structures were introduced [13, 14].
One of the steps in the synthesis of enoxa-
parin is the purification stage, which is a very
important in achieving the equivalence with the
original Clexane® and Lovenox® (Sanofi-Aventis).
Purification of the substance involves decolori-
zation of enoxaparin sodium, pH correction, eli-
mination of degradation products after depoly-
merization, and correction of the molecular com-
position. There are many different methods for
the purification of enoxaparin, for example, lyo-
philization of enoxaparin sodium solution, deco-
lorization of the solution with hydrogen peroxide,
ISSN 2308-8303 (Print) / 2518-1548 (Online) 40
Журнал органічної та фармацевтичної хімії 2023, 21 (3)
followed by reprecipitation using carbon filters,
ion exchange resins, etc. [15]. In the experiment
described in this article, the solution reprecipita-
tion was used as a purification method. It is also
known that the amount of the solvent for repre-
cipitation of enoxaparin affects the number and
distribution of short and long saccharide chains
in the molecule, so we focused on this. Decolori-
zation of the solution is also an important com-
ponent of obtaining API of proper quality, but it
is not a priority of this experiment.
Compliance with the principles of “green” che-
mistry is no less important in the development
of synthetic technologies. “Green” chemistry is
a direction in modern chemistry that consists in
the improvement of technologies regarding the
effective use of the raw material and energy, the
avoidance of toxic and poisonous substances, the
reduction of waste or the repeated use of chemi-
cals and materials [16]. In this study, the E-factor
was chosen as the accent metric for the analysis of
“greenness”. The E-factor is the ratio of the amount
of waste to the amount of a product. All raw ma-
terials used, except water, are included in the
calculation. The higher the value of the E-factor,
the greater the amount of waste [17]. One of the
tasks of this work was also to minimize waste
at the stage of enoxaparin sodium purification,
thereby improving the “greenness” of the synthe-
sis of enoxaparin sodium.
■ The Research Methodology
In the process of planning the experiment for
the purification of crude enoxaparin sodium, an
analysis of the methods described in the litera-
ture was performed.
Thus, the patent [18] describes the following
protocol, which it was decided to use as a basis,
but with a change in the amount of methanol to
observe the effect on the structure of the mole-
cule: “Suspend crude enoxaparin sodium (5 g) in
50 mL of purified water and dissolve. Add 5 g of
sodium chloride and mix. The product is preci-
pitated by adding 150 mL of methanol, filtered,
and dried under vacuum at 55°C for 9 hours,
yielding 4.39 g of enoxaparin sodium”.
The study included tasks outlined below.
1. To purify the substance according to the
parameters selected and to study the effect of the
solvent ratio on the product composition in order
to obtain a substance equivalent to the original
Clexane® and Lovenox®. The following variants
of the ratio of MeOH:H2O were considered: 4:1;
2:1; 1:1.
1.1. First, it was decided to conduct a test ex-
periment to understand the general trend of the
effect of the solvent ratio on the composition and
distribution of low- and high-molecular fractions
of enoxaparin sodium. Thus, crude enoxaparin
sodium synthesized under the so-called “stan-
dard conditions” [11] (base/ester ratio – 0.07; the
reaction mass temperature – 62ºС; the reaction
time – 1 hour) was purified in two ways with the
following solvent ratios:
• МеOH:H2O = 4:1 (D475);
• МеOH:H2O = 1:1 (D478).
1.2. The next step was to analyze the results
obtained for more accurate processing of the pu-
rification stage, including reprecipitation of samp-
les of crude enoxaparin synthesized according to
the optimized parameters of the depolymeriza-
tion stage [11] (alkali/benzyl ester of the hepa-
rin ratio – 0.06; temperature – 57°C, the holding
time of the reaction mixture – 1.5 (D492 and D493)
and 2 hours (D494 and D495) in the ratio of:
• МеOH:H2O = 2:1 (D492 and D494);
• МеOH:H2O = 1:1 (D493 and D495).
2. To optimize the method of synthesis and
purification of enoxaparin sodium in view of the
principles of “green” chemistry.
The reprecipitation stage is carried out at at-
mospheric pressure and room temperature, which
does not contradict the principles of “green” che-
mistry. Methanol, which is a poisonous substan-
ce, is used as a precipitating agent. However, it
is worth noting that according to the in-house
guidelines for the selection of solvents of several
pharmaceutical companies, methanol belongs to
the category “to be confirmed” (Table 1) [19].
In addition, the use of methanol is justified
by the possibility of obtaining a crystalline pre-
cipitate, while the use of solvents with less
harmful environmental effects, such as ethanol
or isopropanol, provokes the formation of a fine-
ly dispersed suspension, which makes it impos-
sible to isolate the precipitate of the substance.
One of the most important indicators of “green”
chemistry is the E-factor, which is a method of
measuring and regulating the amount of waste.
Table 1. Generalized comparison of solvent ratings
Solvent Astra Zeneka GCI-PR GlaxoSmithKlein Pfizer Sanofi Total
MeOH 19 14 14 Preferably Recommended To be confirmed
ISSN 2308-8303 (Print) / 2518-1548 (Online) 41
Journal of Organic and Pharmaceutical Chemistry 2023, 21 (3)
The E-factor is the actual amount of waste de-
fined as “everything except the desired product”
produced per kg of the product, including the loss
of solvents and chemicals used in processing [20].
In pharmaceutical production, solvents account
for 80 – 90 % of the total mass of non-aqueous
material used, most of the waste generated, and
75 – 80 % of the environmental impact of the life
cycle, creating the need for solvent regeneration
with the subsequent reuse. Therefore, the green-
ing of this stage was carried out due to the re-
generation of methanol, which affected the re-
duction of the amount of waste, and, as a result,
a decrease in the E-factor indicator.
3. After analyzing the results of the experi-
mental studies, to make corrections in the me-
thod of the enoxaparin sodium synthesis.
■ Results and discussion
As mentioned earlier, the aim of this work
was to study the effect of solvents on the compo-
sition of the enoxaparin substance. The experi-
ment was conditionally divided into two stages
described below.
1. For the study, we chose samples of crude
enoxaparin sodium that were processed accord-
ing to “standard” non-optimized technological pa-
rameters using the methodology in the patent [12]
and those samples that were processed according
to optimized parameters. Crude enoxaparin
sodium obtained under the so-called “standard
conditions” was purified by reprecipitation of
an aqueous solution of enoxaparin in methanol.
The ratios of water and methanol for reprecipi-
tation were chosen rather roughly, 4:1 and 1:1,
for the initial assessment of the effect of solvents
on the distribution of saccharide fractions with
different molecular weights:
• МеOH:H2O = 4:1 (D475);
• МеOH:H2O = 1:1 (D478).
The samples obtained were analyzed accord-
ing to the internal specification corresponding
to the pharmacopoeial monograph (Table 2).
To quantify the effect of the solvent ratio on the
composite product, enoxaparin sodium was ana-
lyzed by the method of two-dimensional NMR spect-
roscopy (HSQC) (Table 3). In particular, the analy-
sis of the distribution of oligosaccharide fractions
was carried out by the SEC method (Figure 1).
According to Table 2, sample D475 does not
meet the requirements of the specification by the
“Identification” indicator.
According to a more detailed 2D-NMR analy-
sis, both samples (D475 and D478) represent a
high degree of depolymerization, which is eviden-
ced by the low values of normal reduced ANSaRed,
MNSaRed residues and high values of 1,6-anhydro
ANS/MNS, respectively (Table 3). This is the re-
sult of the technological parameters of the depo-
lymerization reaction, which obviously needed to
be corrected.
Table 2. The results of the analysis of samples of purified enoxaparin sodium with varying solvent ratios according to the specification
of JSC Farmak
Parameter Requirements D475 D478
Description A white or almost white powder or crystals meets meets
Solubility Very soluble in water meets meets
Loss on drying, % Not more than 10.0 % 8.59 7.52
pH 6.2 – 7.7 6.31 8.09
Sodium 11.3 – 13.5 12.4 13.2
Specific absorption 14.0 – 20.0 18.5 17.1
Residual amounts of organic
solvents, ppm
methanol – not more than 0.3 % (3000 ppm) 343 13421
methylene chloride – not more than 0.06 % (600 ppm) 0 0
Nitrogen, % 1.5 – 2.5 1.8 1.8
Molar ratio of sulfate ions to
carboxylate ions not less than 1.8 2.92 5.38
Quantitative analysis
factor Xa activity
90 EU – 125 EU 107.9 104.5
factor IIa activity
20.0 EU – 35.0 EU 28.1 27.8
factors Xa/IIa activity ratio
3.3 – 5.3 3.8 3.8
Identification (the average
relative molecular weight and
molecular weight distribution)
3800 – 5000 Da 3978 4239
<2000 Da 12.0 – 20.0 % 21.9 17.8
2000 – 8000 Da 68.0 – 82.0 % 71.1 73.7
Identification (the content of
1,6-anhydro derivatives) 15 – 25 % 23.3 21.8
ISSN 2308-8303 (Print) / 2518-1548 (Online) 42
Журнал органічної та фармацевтичної хімії 2023, 21 (3)
a
b
c
Figure 1. Distribution profiles of oligosaccharide fractions of treated samples of enoxaparin compared to Clexane®: (a) distribution
of oligosaccharide fractions of Clexane® (blue) and crude enoxaparin D466 (pink); (b) distribution of oligosaccharide fractions of crude
enoxaparin (green), purified enoxaparin D475 (pink) and D478 (black); (c) distribution of oligosaccharide fractions of Clexane® (blue)
and purified enoxaparin D478 (pink)
ISSN 2308-8303 (Print) / 2518-1548 (Online) 43
Journal of Organic and Pharmaceutical Chemistry 2023, 21 (3)
The profiles of the distribution of fractions
obtained in the samples synthesized demonstrate
the dynamics of the distribution of the molecu-
lar weight depending on the type of the sample.
Thus, crude enoxaparin sodium (D466, Figure 1a)
coincides with the profile of the originator in the
area of high-molecular fragments, while low-mole-
cular residues remain overestimated compared
to Clexane®. Reprecipitation with the use of the
solvent ratio of MeOH:H2O = 4:1 (D475, Figure
1b) did not give the expected result in reducing
low-molecular-weight particles, but the ratio of
MeOH:H2O = 1:1 (D478, Figure 1c), on the con-
trary, showed a significant effect in this area,
making this sample as close as possible to the
originator.
According to the analyses conducted, the
MeOH:H2O 4:1 ratio option can be immediately
excluded from the study, while the 1:1 solvent
ratio experiment was repeated after adjusting
the parameters of the chemical β-elimination
stage to create a kind of the correct molecular
framework.
2. According to the results of the experiment
on setting the technological parameters of the
depolymerization stage, the optimal parameters
of the process were determined:
• the ratio of “alkali/benzyl ester of hepa-
rin” – 0.06;
• reaction temperature – 57oC;
• the reaction time – an interval of 1.5 – 2 hours.
Samples of crude enoxaparin obtained ac-
cording to these parameters were reprecipitated
with the solvent ratio of:
• МеOH:H2O = 2:1 (D492 and D494);
• МеOH:H2O = 1:1 (D493 and D495).
The samples obtained were also analyzed ac-
cording to the specification developed based on
the pharmacopoeial monograph. The results and
comparison of sample indicators are given in
Table 4. For these samples, the compositional
analysis by the HSQC method (Table 5) and the
molecular weight distribution by the SEC meth-
od (Figure 2) were also determined.
The samples of purified enoxaparin obtained
were analyzed according to the specifications of JSC
Farmak. These samples, as expected, demonstrated
compliance with the regulated requirements of the
monograph in terms of “Identification” (the average
relative molecular weight and molecular weight
Table 3. The results of the analysis of samples of purified enoxaparin sodium with varying amounts of methanol for precipitation
by the HSQC method (2D-NMR)
Crude enoxaparin
sodium MeOH:H2O 4:1 MeOH:H2O 1:1 Clexane
Amines D466 D475 D478 min max
ANS,6xaRed 8.4 7.5 7.4 7.8 9.0
ANS,6XbRed 0.9 1.2 0.8 1.0 1.2
ANAc,6xaRed 0.4 0.4 0.4 0.3 0.4
1,6anANS 3.3 3.3 3.0 2.0 2.3
1,6anMNS 3.9 3.5 3.2 2.4 2.5
MNS,6XaRed 2.4 2.2 2.0 2.6 3.0
%A6S 78.7 80.2 80.8 81.8 82.9
Uronic acid
ΔU42S 20.4 19.0 17.5 17.3 18.1
ΔU4 1.9 1.7 1.5 1.1 1.2
Epox 1.3 1.4 1.4 0.2 0.6
GalA 1.8 1.9 1.9 1.2 1.8
ΔU42S/ΔU 10.7 11.3 11.5 15.7 15.1
Notes:
Abbreviation Stands for
ANS,6X-αRed reducing N-sulfated-α-D-glucosamine
ANS,6XβRed reducing N-sulfated-β-D-glucosamine
ANAc,6X-αRed reducing N-acetyl α-D-glucosamine
1,6anANS 2-amino-1,6-anhydro-2-deoxy-β-D-glucopyranose
1,6anMNS 2-amino-1,6-anhydro-2-deoxy-β-D-mannopyranose
MNS,6XαRed reducing N-sulfated-α-D-mannosamine
%A6S N-sulfated/acetylated 6-O-sulfated α-D-glucosamine/mannosamine percent
ΔU42S 2-O-sulfo-4-deoxy-α-L-threo-hex-4-enopyranosil uronic acid
ΔU4 4-deoxy-α-L-threo-hex-4-enopyranosil uronic acid
epox epoxide residue
GalA galacturonic acid
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Журнал органічної та фармацевтичної хімії 2023, 21 (3)
distribution). According to the results of the com-
positional analysis, we observe a tendency to de-
crease the number of residues at the reducing ends
of the molecule – ANS/MNSred, 1,6anMNS/ANS
and structures at the non-reducing ends of the
molecule – ΔU42S, ΔU4, which, however, still does
not coincide with the variation ranges of Clexane®.
The analysis of the molecular weight distri-
bution shows that the samples D492 and D494
obtained with the ratio of MeOH:H2O=2:1 have
a larger number of residues with a low-molecu-
lar weight than Clexane®. Similarly, high-mole-
cular-weight fragments are more common in D492
and D494 than in Clexane® (Figure 2).
Table 4. The results of the analysis of samples of purified enoxaparin sodium with a change in the ratio of solvents according
to the specification of JSC Farmak
Parameter Requirements
MeOH:H2O 2:1 MeOH:H2O 1:1
D492 D494 D493 D495
Description A white or almost white powder
or crystals meets meets meets meets
Solubility Very soluble in water meets meets meets meets
Loss on drying, % Not more than 10.0 % 6.13 8.71 7.30 6.76
pH 6.2 – 7.7 7.72 7.23 6.93 7.06
Sodium 11.3 – 13.5 11.3 11.7 11.8 11.6
Specific absorption 14.0 – 20.0 17.3 17.1 15.2 15.5
Residual amounts of organic
solvents, ppm
methanol – not more than 0.3 %
(3000 ppm) 583 150 1110 5407
methylene chloride – not more
than 0.06 % (600 ppm) 0 0 0 0
Nitrogen, % 1.5 – 2.5 1.9 2.0 1.9 2.0
Molar ratio of sulfate ions to
carboxylate ions not less than 1.8 2.4 3.5 2.3 2.9
Quantitative analysis
factor Xa activity
90 EU – 125 EU 109.1 101.6 113.7 103.6
factor IIa activity
20,0 EU – 35,0 EU 29.7 29.7 36.1 35.0
factors Xa/IIa activity ratio
3.3 – 5.3 3.7 3.4 3.1 3.0
Identification (the average
relative molecular weight and
molecular weight distribution)
3800 – 5000 Da 4553 4625 4880 4905
< 2000 Da
12.0 % – 20.0 % 18.1 17.0 12.5 12.5
2000-8000 Da
68.0 % – 82.0 % 70.2 71.0 74.5 74.2
Identification (the content of
1,6-anhydroderivatives) 15 – 25 % 17.2 19.9 15.6 18.7
Table 5. The results of the analysis of samples of purified enoxaparin sodium with varying amounts of methanol for precipitation
by the HSQC method (2D-NMR)
Crude enoxaparin
MeOH:H2O 2:1 MeOH:H2O 1:1 Clexane
1.5 h 2 h
Amines D484 D485 D492 D494 D493 D495 min max
ANS,6xaRed 10.1 9.6 9.8 9.0 8.5 8.0 7.8 9.0
ANS,6XbRed 1.3 1.2 1.0 1.2 1.2 1.1 1.0 1.2
ANAc,6xaRed 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.4
1,6anANS 2.2 2.5 2.0 2.1 1.7 1.9 2.0 2.3
1,6anMNS 2.5 2.7 2.2 2.3 1.9 2.1 2.4 2.5
MNS,6XaRed 2.9 2.7 2.7 2.4 2.4 2.2 2.6 3.0
%A6S 80.4 80.0 80.8 80.3 81.8 81.4 81.8 82.9
Uronic acid
ΔU42S 19.2 19.2 18.1 17.6 15.7 15.7 17.3 18.1
ΔU4 1.7 1.7 1.5 1.5 1.2 1.2 1.1 1.2
Epox 0.9 0.9 0.9 1.0 0.9 1.0 0.2 0.6
GalA 2.0 1.9 1.9 1.9 1.9 1.8 1.2 1.8
ΔU42S / ΔU 11.3 11.1 11.9 11.8 13.3 12.7 15.7 15.1
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Journal of Organic and Pharmaceutical Chemistry 2023, 21 (3)
d
e
f
Figure 2. Distribution profiles of oligosaccharide fractions of treated samples of purified enoxaparin compared to Clexane®:
(d) distribution of oligosaccharide fractions of purified enoxaparin D492 (yellow) and D493 (blue); (e) distribution of oligosaccharide
fractions of purified enoxaparin D494 (blue) and D495 (red); (f) distribution of oligosaccharide fractions of Clexane® (black) and D492
(yellow); (g) distribution of oligosaccharide fractions of Clexane® (black) and D493 (blue); (h) distribution of oligosaccharide fractions
of Clexane® (black) and D494 (blue); (i) distribution of oligosaccharide fractions Clexane® (black) and D495 (red) (see on the next page)
ISSN 2308-8303 (Print) / 2518-1548 (Online) 46
Журнал органічної та фармацевтичної хімії 2023, 21 (3)
g
h
i
Figure 2. Distribution profiles of oligosaccharide fractions of treated samples of purified enoxaparin compared to Clexane®:
(d) distribution of oligosaccharide fractions of purified enoxaparin D492 (yellow) and D493 (blue); (e) distribution of oligosaccharide
fractions of purified enoxaparin D494 (blue) and D495 (red); (f) distribution of oligosaccharide fractions of Clexane® (black) and D492
(yellow); (g) distribution of oligosaccharide fractions of Clexane® (black) and D493 (blue); (h) distribution of oligosaccharide fractions
of Clexane® (black) and D494 (blue); (i) distribution of oligosaccharide fractions Clexane® (black) and D495 (red)
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Journal of Organic and Pharmaceutical Chemistry 2023, 21 (3)
Samples D493 and D495 (Figures 2g,i) show
a profile similar to Clexane® in the range of
short oligomers, indicating that the ratio of
MeOH:H2O=1:1 allows better control of the num-
ber of low molecular weight oligomers. However,
the intensity of the high-molecular range is higher
than that of Clexane®. A decrease in the number
of short particles, depending on the amount of
methanol, shifts the molecular weight distribu-
tion towards high-molecular weight.
The methanol regeneration was envisaged
as the greening stage of the synthesis. The re-
generation yield was 70 %. The calculation of the
E-factor considering the regeneration is shown
in Table 6. The E-factor value obtained without
the methanol regeneration is 14. Recalculation
of the E-factor considering the methanol regen-
eration is 5.25.
■ Conclusions
In this experimental study, it was possible to
clearly demonstrate the effect of the ratio of sol-
vents at the stage of purification of crude enoxa-
parin on the number and distribution of oligo-
saccharide fractions in the molecule. Thus, it
has been found that an increase in the amount
of methanol for the reprecipitation of enoxapa-
rin provokes a shift in the profile of the molecu-
lar weight distribution towards low molecular
weight oligosaccharides, respectively, a smaller
amount of methanol allows obtaining the profile
closest to the originator in the area of low-mole-
cular-weight residues. The ratio of MeOH:H2O =
1:1 makes it possible to obtain samples that are
better comparable in terms of the composition
to the ranges of Clexane®, except for the termi-
nal residues. However, during the study, it was
found that with the reduction of low-molecular-
weight residues, the intensity in the area of high-
molecular-weight oligosaccharides increased.
Summarizing the results obtained, it can be
concluded that the ratio of MeOH:H2O = 1:1 is
acceptable for obtaining a substance close to the
original one. As an indicator of the effectiveness
of the method of the purification stage of enoxa-
parin sodium in view of the principles of “green”
chemistry, there is an E-factor reduced from 14
to 5.25 by the methanol regeneration.
■ Experimental part
This study was conducted during 2019 – 2021.
The treated samples of purified enoxaparin
sodium were analyzed according to the internal
specification developed based on the pharmaco-
poeial monograph. For detailed structural cha-
racterization of enoxaparin sodium samples ob-
tained under different conditions, the analysis
was performed by specialists of the Ronzoni In-
stitute (Italy) using the methods of 2D-NMR
(heteronuclear single quantum coherence spec-
troscopy) and size exclusion chromatography
(SEC). The results of the analysis were compa-
red with the results of the analysis of the origi-
nal Clexane® referring to the database formed
by the Ronzoni Institute.
Clexane® from Sanofi-Aventis was obtained
from commercial suppliers.
All samples were analyzed before the expira-
tion date.
The pH test was determined on a Mettler To-
ledo Seven compact S220 pH meter (Switzerland)
(Ph. Eur. 2.2.3), the analysis of loss on drying
was performed on a Pol-Eko Aparatura slw 53
(Ph. Eur. 2.2.32); nitrogen was analyzed on a Va-
podest VAP 30s Gerhardt GmbH Distillation
System (Ph. Eur. 2.5.9); the analysis of residual
amounts of organic solvents was carried out by
the head-space gas chromatography method on
an Agilent GC 7890B chromatograph (USA), co-
lumn DB-624, 60m×0.32mm, with a layer thick-
ness of 1.8 μm (Ph. Eur. 2.2.28, 2.2.46); specific
absorption was measured on a Mettler Toledo UV-5
spectrophotometer (Ph. Eur. 2.2.25); identifica-
tion (the average relative molecular weight and
Table 6. Calculation of the E-factor of the purification stage of crude enoxaparin sodium considering the solvent regeneration
Materials
Quantity of materials, kg
Product
yield, kg E-factor
Е-factor that takes
into account the
regeneration
Without the solvent
regeneration
With the solvent
regeneration
Crude enoxaparin sodium 0.1 0.1
0.08 14 5.25
Sodium chloride 0.1 0.1
Methanol 1.0 0.3
1.2 0.5
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Журнал органічної та фармацевтичної хімії 2023, 21 (3)
molecular weight distribution) was performed
on a Shimadzu chromatograph (Japan), column
Х_TSKgel G2000SW (300mm×7.8mm×5μm) with
a Viscotec 305 detector, Malvern Instruments LTD
(England) (Ph. Eur. 2.2.30). The content of 1,6-an-
hydro derivatives was measured by the LC me-
thod on a Metrohm chromatograph (Ph. Eur. 2.2.29).
The molar ratio of sulfate ions to carboxylate ions
was measured on a Seven Compact S230 con-
ductometer (Switzerland) (Ph. Eur. 2.2.38).
■ Acknowledgements
The authors would like to thank Marco
Guerrini and Cristina Gardini from The Centro
Alta Tecnologia Istituto di Ricerche Chimiche e
Biochimiche G. Ronzoni (Milan, Italy) for their
excellent skills and assistance in the enoxapa-
rin sodium project in JSC Farmak and the study
of the samples by the methods of NMR and size-
exclusion chromatography.
■ References
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Journal of Organic and Pharmaceutical Chemistry 2023, 21 (3)
Information about the authors:
Yuliia V. Bovsunovska (corresponding author), Ph.D. student of the Pharmaceutical Chemistry Department, National University
of Pharmacy of the Ministry of Health of Ukraine; Engineer-technologist, JSC Farmak; https://orcid.org/0000-0001-6525-5520;
e-mail for correspondence: juliabovsu@gmail.com.
Vitalii V. Rudiuk, Head of the API Synthesis Laboratory, JSC Farmak; https://orcid.org/0000-0003-3440-1139.
Nataliia V. Harna, Ph.D. in Pharmacy, Associate Professor of the Pharmaceutical Chemistry Department, National University
of Pharmacy of the Ministry of Health of Ukraine; https://orcid.org/0000-0003-2918-4985.
Olha S. Holovchenko, Ph.D. in Pharmacy, Associate Professor of the Pharmaceutical Chemistry Department, National University
of Pharmacy of the Ministry of Health of Ukraine; https://orcid.org/0000-0002-5252-5517.
Victoriya A. Georgiyants, D.Sc. in Pharmacy, Professor, Head of the Department of Pharmaceutical Chemistry, National University
of Pharmacy of the Ministry of Health of Ukraine; http://orcid.org/0000-0001-8794-8010.
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| id | oai:ojs.journals.uran.ua:article-290670 |
| institution | Journal of Organic and Pharmaceutical Chemistry |
| issn | 2518-1548 |
| keywords_txt_mv | |
| language | English |
| last_indexed | 2026-08-24T01:00:48Z |
| publishDate | 2023 |
| publisher | National University of Pharmacy |
| record_format | ojs |
| resource_txt_mv | ophcjnupheduua/46/3c391d7a0e8a4a4da978c38b18a9dc46.pdf |
| spelling | oai:ojs.journals.uran.ua:article-2906702026-08-23T18:59:42Z Obtaining the Enoxaparin Sodium Substance Equivalent to the Original Clexane® and Lovenox®. The Selection of Technological Parameters and Optimization of the “Greenness” of the Purification Stage Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення Bovsunovska, Yuliia V. Rudiuk, Vitalii V. Harna, Nataliia V. Holovchenko, Olha S. Georgiyants, Victoriya A. enoxaparin sodium low-molecular-weight heparin technological parameters compositional analysis HSQC size-exclusion chromatography green chemistry E-factor solvent regeneration еноксапарин натрію низькомолекулярний гепарин технологічні параметри композиційний аналіз HSQC ексклюзійна хроматографія зелена хімія Е-фактор регенерація розчинника The aim of the study was to adjust and optimize the purification stage of crude enoxaparin sodium to obtain a substance equivalent to the original drugs Clexane® and Lovenox® according to the criteria specified by the FDA. The purification stage involves the reprecipitation of crude enoxaparin in methanol. Determining the ratio of solvents required for the reprecipitation is important for studying the correlation between the experimental conditions of the technological process and the structural characteristics of enoxaparin samples. In the study, the method of purification of enoxaparin sodium described in the patent was assessed, and the following variations of the MeOH:H2O solvent ratio were selected – 4:1; 2:1; 1:1. The obtained samples of enoxaparin sodium were analyzed according to the in-house specification developed on the basis of the pharmacopoeial monograph, as well as by non-pharmacopoeial methods, such as two-dimensional NMR spectroscopy (HSQC) and size exclusion chromatography (SEC) for detailed characterization of the molecule. Strategies of greening of the enoxaparin sodium purification stage by reducing the E-factor were also considered in the study. Considering the principles of “green” chemistry, the method of purification of crude enoxaparin sodium was optimized by the solvent regeneration. It was experimentally possible to demonstrate the effect of the solvent ratio at the stage of purification of crude enoxaparin on the composition, as well as on the number and distribution of oligosaccharide fractions in the molecule. Based on the results of the study, it can be concluded that the ratio of MeOH:H2O=1:1 allows obtaining samples that are closest to Clexane® and Lovenox® in terms of the molecular weight distribution profile and the composition profile. The E-factor was also reduced from 14 to 5.25 by solvent regeneration. Метою роботи було налаштувати та оптимізувати стадію очищення технічного еноксапарину натрію для отримання субстанції, еквівалентної оригінальним препаратам Clexane® та Lovenox® за критеріями, окресленими FDA. Стадія очищення передбачає переосадження неочищеного еноксапарину із метанолу. Визначення необхідного співвідношення розчинників для переосадження є важливим для дослідження кореляції між експериментальними умовами технологічного процесу та структурними характеристиками зразків еноксапарину. У дослідженні було оцінено спосіб очищення еноксапарину натрію, описаний у патенті, і обрано такі варіанти співвідношення розчинників MeOH:H2O – 4:1; 2:1; 1:1. Отримані зразки еноксапарину натрію аналізували відповідно до внутрішньої специфікації, розробленої на основі фармакопейної монографії, а також за допомогою нефармакопейних методів, таких, як двовимірна ЯМР-спектроскопія (HSQC) та ексклюзійна хроматографія (SEC) для детальної характеристики. молекули. У дослідженні також розглядали стратегії екологізації етапу очищення еноксапарину натрію шляхом зниження Е-фактора. З огляду на принципи «зеленої» хімії метод очищення неочищеного еноксапарину натрію було оптимізовано шляхом регенерації розчинника. Експериментально вдалося продемонструвати вплив співвідношення розчинників на стадії очищення неочищеного еноксапарину на склад, а також на кількість і розподіл фракцій олігосахаридів у молекулі. За результатами дослідження можна зробити висновок, що співвідношення MeOH:H2O = 1:1 дозволяє отримати зразки, які за профілем молекулярно-масового розподілу та профілем складу найбільш наближені до Clexane® та Lovenox®. Е-коефіцієнт також було знижено з 14 до 5,25 шляхом регенерації розчинника. National University of Pharmacy 2023-11-07 Article Article application/pdf https://ophcj.nuph.edu.ua/article/view/290670 10.24959/ophcj.23.290670 Journal of Organic and Pharmaceutical Chemistry; Vol. 21 No. 3 (2023); 38-49 Журнал органической и фармацевтической химии; Том 21 № 3 (2023); 38-49 Журнал органічної та фармацевтичної хімії; Том 21 № 3 (2023); 38-49 2518-1548 2308-8303 en https://ophcj.nuph.edu.ua/article/view/290670/285060 Copyright (c) 2023 Yuliia V. Bovsunovska, Vitalii V. Rudiuk, Nataliia V. Harna, Olha S. Holovchenko, Victoriya A. Georgiyants http://creativecommons.org/licenses/by/4.0 |
| spellingShingle | еноксапарин натрію низькомолекулярний гепарин технологічні параметри композиційний аналіз HSQC ексклюзійна хроматографія зелена хімія Е-фактор регенерація розчинника Bovsunovska, Yuliia V. Rudiuk, Vitalii V. Harna, Nataliia V. Holovchenko, Olha S. Georgiyants, Victoriya A. Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення |
| title | Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення |
| title_alt | Obtaining the Enoxaparin Sodium Substance Equivalent to the Original Clexane® and Lovenox®. The Selection of Technological Parameters and Optimization of the “Greenness” of the Purification Stage |
| title_full | Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення |
| title_fullStr | Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення |
| title_full_unstemmed | Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення |
| title_short | Одержання субстанції еноксапарину натрію, еквівалентної оригінальним Clexane® та Lovenox®. Підбір технологічних параметрів та оптимізація «зеленості» стадії очищення |
| title_sort | одержання субстанції еноксапарину натрію, еквівалентної оригінальним clexane® та lovenox®. підбір технологічних параметрів та оптимізація «зеленості» стадії очищення |
| topic | еноксапарин натрію низькомолекулярний гепарин технологічні параметри композиційний аналіз HSQC ексклюзійна хроматографія зелена хімія Е-фактор регенерація розчинника |
| topic_facet | enoxaparin sodium low-molecular-weight heparin technological parameters compositional analysis HSQC size-exclusion chromatography green chemistry E-factor solvent regeneration еноксапарин натрію низькомолекулярний гепарин технологічні параметри композиційний аналіз HSQC ексклюзійна хроматографія зелена хімія Е-фактор регенерація розчинника |
| url | https://ophcj.nuph.edu.ua/article/view/290670 |
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