Experimental Testing of a Size-Exclusion Chromatography Method Used for Evaluation of Molecular Parameters of Equine Anti-Ebola Immunoglobulin

Haemorrhagic fever caused by the Ebola virus is a highly hazardous infectious disease with a mortality rate of 50– 90 %. Heterologous immunoglobulins with a high virus-neutralizing titer are an important element of the WHO-endorsed set of measures for emergency prevention and treatment of the disease. Specific activity of these products is largely determined by their fractional composition, and, in particular, by molecular mass distribution (MMD). The size-exclusion-high-performance liquid chromatography (SEC-HPLC) has traditionally been used for determination of the MMD of the target protein in human immunoglobulin-based products. The use of this method for evaluation of molecular parameters of heterologous immunoglobulin requires confirmation of its specificity, accuracy and precision, and establishment of the chromatographic system suitability criteria in the context of a new test object.The aim of the study was to test the applicability of the SEC-HPLC method to the assessment of molecular parameters of anti-Ebola immunoglobulin derived from horse serum.Materials and methods: three batches of purified equine anti-Ebola immunoglobulin were used in the study. Normal equine and human immunoglobulins of the IgG isotype were used as reference standards. The HPLC test procedures described in the European Pharmacopoeia 9.6 and State Pharmacopoeia of the Russian Federation, 14th ed., were used for determination of monomers and other immunoglobulin fractions. An Agilent 1260 Infinity (Agilent, USA) HPLC system with a diode array detector and an Agilent Bio SEC-3 HPLC column were used for quality evaluation of the tested products.Results: the resolution factor between IgG monomer and dimer peaks (1.69 and 2.10), and the chromatographic column efficiency (>2000) make it possible to use the SEC-HPLC system for evaluation of molecular parameters of heterologous immunoglobulin. The study demonstrated reproducibility of the test procedure.Conclusions: the study confirmed the applicability of the SEC-HPLC procedure for evaluation of molecular parameters of anti-Ebola immunoglobulin derived from horse serum. It demonstrated the compliance of the purified immunoglobulin to the national and international quality requirements in terms of «Molecular parameters».

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Determination of Vancomycin B and Vancomycin Impurities by Liquid Chromatography

The Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products ◽

10.30895/1991-2919-2021-11-4-246-254 ◽

2021 ◽

Vol 11 (4) ◽

pp. 246-254

Author(s):

S. I. Kuleshova ◽

E. P. Simonova ◽

O. N. Vysochanskaya

Keyword(s):

Liquid Chromatography ◽

Chromatographic Separation ◽

Separation Efficiency ◽

Test Procedure ◽

Testing Time ◽

Test Procedures ◽

British Pharmacopoeia ◽

Related Impurities ◽

Vancomycin Hydrochloride

The preferred test methods for control of product-related impurities in medicinal products are high-performance liquid chromatography (HPLC) with a fine sorbent, and ultra-performance liquid chromatography (UPLC), which allow for better chromatographic separation of active substances and related impurities, reduction of time costs, and saving of material resources. The aim of the study was to develop HPLC and UPLC test procedures and assess the chromatographic separation capacity and efficiency in order to improve determination of the main vancomycin component and related impurities. Materials and methods: vancomycin hydrochloride lyophilisate for oral solution and solution for injection, and vancomycin hydrochloride reference standard (USP RS) were used as test objects. Agilent 1290 Infinity liquid chromatography system, and Chromolith® Performance RP-18e, Kinetex C18, Nucleodur C18 Isis, Zorbax RRHD Eclipse Plus C18, and LiChrospher® RP-18 columns were used for the testing. Results: HPLC analysis using a Chromolith® column (100×4.6 mm) reduces the testing time by 10 minutes compared to the USP test procedure, and by 15 minutes compared to the British Pharmacopoeia procedure. The proposed test procedure requires less eluent and increases chromatographic separation efficiency. UPLC analysis using a Kinetex C18 column (50×4.6 mm, 2.6 μm) made it possible to reduce the testing time by two thirds compared to the British Pharmacopoeia procedure. The use of isocratic elution greatly simplified the testing. The testing time under the proposed chromatographic conditions was 10 minutes. Conclusions: the selected HPLC and UPLC test conditions made it possible to significantly reduce the time of testing, minimise the use of expensive reagents, and increase efficiency of chromatographic separation in the determination of vancomycin impurities and the main component Vancomycin B.

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Detection of Pyrogens in Hormonal Implants Using the LAL Test

The Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products ◽

10.30895/1991-2919-2021-11-2-115-120 ◽

2021 ◽

Vol 11 (2) ◽

pp. 115-120

Author(s):

N. P. Neugodova ◽

O. V. Shapovalova ◽

G. A. Sapozhnikova ◽

E. O. Stepanyuk

Keyword(s):

Dimethyl Sulfoxide ◽

Dosage Form ◽

Test Procedure ◽

Water Extract ◽

Test Procedures ◽

Implant Surface ◽

Bacterial Endotoxins ◽

Kinetic Test ◽

Second Procedure

The State Pharmacopoeia of the Russian Federation, 14th edition states that implants are a sterile dosage form, and have to be tested for pyrogens. However, it does not provide details on how the test should be performed for this dosage form.The aim of the study was to develop a LAL test procedure for detection of bacterial endotoxins (BE) in implants, using the example of a goserelin product.Materials and methods: BE extraction from the implant surface into an aqueous medium was performed with subsequent BE detection in the extract by turbidimetric kinetic test. The implant was then dissolved in dimethyl sulfoxide, and the obtained goserelin solution was tested for BEs using the gel-clot test.Results: the analysis of the Russian and foreign pharmacopoeial approaches to pyrogenic substance detection in hormonal implants helped to develop two sample preparation procedures for determination of BE content (in the extract and the implant solution). It was demonstrated that the BE content in the water extract did not exceed 0.01 EU/mL and was less than 0.07 EU per implant. The BE content in the implant solution was less than 8.3 EU per 1 mg of goserelin, which is almost eleven-fold lower than the theoretically-derived limit.Conclusions: the authors developed two test procedures for BE detection in hormonal implants using the LAL test, which could be included in manufacturers’ product files. The first procedure involves testing of the water extract from the implant surface and establishes the BE limit of no more than 20 EU/product. The second procedure involves complete dissolution of the implant in dimethyl sulfoxide and establishes the limit of not more than 97.22 EU per 1 mg of goserelin.

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Current Approaches to Valerian Tincture Standardisation in Terms of Assay

The Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products ◽

10.30895/1991-2919-2019-9-4-265-271 ◽

2019 ◽

Vol 9 (4) ◽

pp. 265-271

Author(s):

N. P. Antonova ◽

E. P. Shefer ◽

A. M. Kalinin ◽

N. E. Semenova ◽

S. S. Prokhvatilova ◽

...

Keyword(s):

Quantitative Analysis ◽

Quantitative Determination ◽

High Performance ◽

Test Procedure ◽

Total Content ◽

Reference Standard ◽

Active Ingredients ◽

Test Procedures ◽

Valerenic Acid

The quality control of the «Valerian rhizome and roots» herbal substance is carried out using high performance liquid chromatography (HPLC) according to the State Pharmacopoeia of the Russian Federation, XIV edition. The quantitative analysis of the active ingredients in valerian tincture is performed using a non-specific and non-selective spectrophotometric method. Therefore, it is important to introduce in Russia a more modern test procedure for quantitative determination of active ingredients in valerian tincture.The aim of the study was to develop a selective and sensitive HPLC procedure for quantitative determination of the total content of sesquiterpenic acids, expressed as valerenic acid, for the purpose of valerian tincture standardisation.Materials and methods: valerian tincture samples produced by seven Russian manufacturers were used as test samples, and valerenic acid was used as the reference standard. The quantitative analysis of the active ingredients was performed by two methods: spectrophotometry at 512 nm following the reaction of valerenic acid ethylester with hydroxyalamine and ferric chloride, and by HPLC using a Nucleosil C18 column, 125×4.6 mm, 5 µm particle size, in gradient elution mode, with detection at 220 nm.Results: the spectrophotometric technique was shown to be insufficiently specific. The authors of the study validated the chromatographic test procedure, established system suitability criteria, and compared the results obtained by the two test procedures. They also determined a tentative standard of the total content of sesquiterpenic acids, expressed as valerenic acid, obtained by HPLC.Conclusions: the HPLC assay developed for quantitative determination of active ingredients in valerian tincture is more specific as compared to the spectrophotometric technique, as the sum of the peaks of valerenic and acetoxyvalerenic acids and the results for the reference standard are taken into account during calculations. The new test procedure is in line with the cross-cutting standardisation principle and can be recommended for inclusion into the draft monograph «Valerian tincture».

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Reliability of Laboratory Tests for the Control of Oral Anticoagulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647716 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 483-491

Author(s):

E. A Loeliger ◽

M. J Boekhout-Mussert ◽

L. P van Halem-Visser ◽

J. D. E Habbema ◽

H de Jonge

Keyword(s):

Human Brain ◽

Laboratory Tests ◽

Test Procedure ◽

Rabbit Brain ◽

Normal Range ◽

Test Procedures ◽

Discrimination Power ◽

Power Of The Test ◽

Assay Procedure ◽

Low Sensitivity

SummaryThe present study concerned the reproducibility of the so-called prothrombin time as assessed with a series of more commonly used modifications of the Quick’s onestage assay procedure, i.e. the British comparative reagent, homemade human brain thromboplastin, Simplastin, Simplastin A, and Thrombotest. All five procedures were tested manually on pooled lyophilized normal and patients’ plasmas. In addition, Simplastin A and Thrombotest were investigated semiautomatically on individual freshly prepared patients’ plasmas. From the results obtained, the following conclusions may be drawn :The reproducibility of results obtained with manual reading on lyophilized plasmas is satisfactory for all five test procedures. For Simplastin, the reproducibility of values in the range of insufficient anticoagulation is relatively low due to the low discrimination power of the test procedure in the near-normal range (so-called low sensitivity of rabbit brain thromboplastins). The reproducibility of Thrombotest excels as a consequence of its particularly easily discerned coagulation endpoint.The reproducibility of Thrombotest, when tested on freshly prepared plasmas using Schnitger’s semiautomatic coagulometer (a fibrinometer-liJce apparatus), is no longer superior to that of Simplastin A.The constant of proportionality between the coagulation times formed with Simplastin A and Thrombotest was estimated at 0.64.Reconstituted Thrombotest is stable for 24 hours when stored at 4° C, whereas reconstituted Simplastin A is not.The Simplastin A method and Thrombotest seem to be equally sensitive to “activation” of blood coagulation upon storage.

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An Enzyme Linked Immunosorbent Assay for Determination of Tissue Plasminogen Activator Applied to Patients with Thromboembolic Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665299 ◽

1983 ◽

Vol 50 (03) ◽

pp. 740-744 ◽

Cited By ~ 139

Author(s):

Nils Bergsdorf ◽

Torbjörn Nilsson ◽

Per Wallén

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Specific Activity ◽

Thromboembolic Disease ◽

Tissue Type ◽

Enzyme Linked Immunosorbent Assay ◽

Type Plasminogen Activator ◽

Tissue Plasminogen ◽

Normal Human

SummaryUtilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme- linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 ± 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tomiquet test, and to 14 ng/ml after strenous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-α2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

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