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Study of a combined biological product with antibacterial and probiotic activity 一种具有抗菌和益生菌活性的复合生物制品的研究
Pub Date : 2023-10-06 DOI: 10.30895/2221-996x-2023-23-3-1-422-430
V. A. Neschislyaev, E. G. Shilova, A. M. Nikolaeva, E. V. Orlova
Scientific relevance . The issue of antimicrobial resistance has acquired global significance, urging the search for and use of alternative antibacterial agents to treat infectious diseases. In particular, this situation prompts wider use of biotechnology-derived medicinal products based on bacteriophages and probiotics, including those of the metabolite type. The bacteriotropic (antimicrobial and probiotic) properties of pharmaceutical compositions based on bacteriophages and metabolites of probiotic bacteria suggest a qualitatively new combined antibacterial effect, as well as effectiveness against microorganisms resistant to antibiotics. Aim . The authors aimed to study the properties of a combined biological product with antibacterial and probiotic effects. Materials and methods . The study focused on a combined biological product consisting of a probiotic agent based on Lactobacillus exometabolites and a complex bacteriophage. The antimicrobial activity evaluation involved the Appelmans method, a disc diffusion method using lawn cultures homologous ( Staphylococcus spp., Pseudomonas aeruginosa , Escherichia coli , Proteus spp., Enterococcus spp., Salmonella spp., and Shigella spp.) and non-homologous ( Acinetobacter baumannii, Klebsiella pneumoniae , and Yersinia enterocolitica ) to the complex bacteriophage, and a bioluminescence inhibition test using the genetically modified indicator strain E. coli lum+. The study used bacterial strains isolated from various clinical samples and biotopes in bacteriological laboratories of healthcare institutions in the Perm Territory in 2019–2022. The probiotic activity was assessed by the stimulating effect on model microorganisms of the normal flora. Results . The Appelmans method showed that the combined biological product had high antimicrobial activity against microorganisms homologous to the complex bacteriophage. The titres calculated for the combined biological product and its complex bacteriophage component were comparable and amounted to 10 –6.6±0.01 and 10 –6.9±0.01 , respectively. The disc diffusion method demonstrated that the combined biological product had a more pronounced antibacterial effect on all tested strains than its individual components. The optical density values obtained with the combined biological product were 1.5 and 2 times higher than the values observed with control samples in Bifidobacterium bifidum 1 and Lactobacillus plantarum 8P-А3 cultures, respectively, which demonstrated the stimulating effect of the product on the normal flora. The study results suggest the compatibility of the phage and probiotic components of the combined biological product, as well as its high antimicrobial activity. Conclusions . The novel combined biological product has high specific and non-specific antimicrobial activity, which consists in the inhibition of pathogenic bacteria growth without affecting the normal flora. The combined biological product broadens the range of medicinal products
科学相关性。抗菌素耐药性问题已具有全球意义,促使人们寻找和使用替代抗菌剂来治疗传染病。特别是,这种情况促使更广泛地使用基于噬菌体和益生菌的生物技术衍生药物,包括代谢物类型的药物。基于益生菌的噬菌体和代谢物的药物组合物的嗜菌性(抗菌和益生菌)特性表明了一种定性的新型联合抗菌效果,以及对抗生素耐药微生物的有效性。的目标。作者旨在研究一种具有抗菌和益生菌作用的复合生物制品的性能。材料和方法。本研究的重点是一种组合生物制品,由一种基于乳酸杆菌外代谢产物的益生菌剂和一种复杂的噬菌体组成。抗菌活性评估包括Appelmans方法,使用与复合噬菌体同源(葡萄球菌、铜绿假单胞菌、大肠杆菌、变形杆菌、肠球菌、沙门氏菌和志贺氏菌)和非同源(鲍曼不动杆菌、肺炎克雷伯菌和小肠结肠炎耶尔森菌)的草坪培养的光盘扩散法,以及使用转基因指示菌株大肠杆菌+进行生物发光抑制试验。该研究使用了2019-2022年从彼尔姆领土医疗机构细菌学实验室的各种临床样本和生物群落中分离出来的细菌菌株。通过对正常菌群模式微生物的刺激作用来评价益生菌活性。结果。Appelmans方法表明,复合生物制品对复合噬菌体同源微生物具有较高的抑菌活性。复合生物制品及其复合噬菌体组分的滴度具有可比性,分别为10 -6.6±0.01和10 -6.9±0.01。圆盘扩散法表明,该组合生物制品对所有被试菌株的抑菌效果比其单独成分更明显。在两歧双歧杆菌1和植物乳杆菌8P-А3培养物中,联合生物制品获得的光密度值分别是对照样品的1.5倍和2倍,证明了该生物制品对正常菌群的刺激作用。研究结果表明,该组合生物制品的噬菌体和益生菌组分具有良好的相容性,且具有较高的抗菌活性。结论。该新型组合生物制品具有高度的特异性和非特异性抗菌活性,即在不影响正常菌群的情况下抑制病原菌的生长。该组合生物制品扩大了具有益生菌和抗菌作用的医药产品的范围,特别是对抗生素耐药的微生物。
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引用次数: 0
Probiotic activity of <i>Bacillus subtilis</i> metabolites in experimentally induced dysbiosis in mice 枯草芽孢杆菌的益生菌活性实验诱导小鼠生态失调的代谢物
Pub Date : 2023-09-25 DOI: 10.30895/2221-996x-2023-23-445
S. А. Lazarev, N. O. Vartanova, A. V. Poddubikov, N. A. Mikhailova
Scientific relevance . A promising option for dysbiosis correction is the use of metabiotics, products based on metabolites of probiotic microorganisms. During fermentation, Bacillus subtilis bacteria (strains 3H and 1719) produce metabolites that exhibit probiotic properties in vitro . These observations in vitro motivate an in vivo investigation of B. subtilis metabolite effects on colonic mucosal microbiota in mice in experimentally induced dysbiosis and an assessment of the potential of B. subtilis metabolites as metabiotics. Aim . The authors aimed to compare the probiotic activity of B. subtilis 3H and B. subtilis 1719 metabolites and a commercial metabiotic in antibiotic-induced dysbiosis in mice. Materials and methods . The authors induced experimental dysbiosis in BALB/c mice weighing 18–20 g by intraperitoneal injection of gentamicin. For subsequent correction, the test groups received sorbent-bound B. subtilis metabolites, and the comparison group received a commercial metabiotic containing B. subtilis metabolites (VKPM B-2335(3)3) via intragastric injection for 21 days. The quantitative and qualitative analysis of colonic mucosal microbiota included microbial culturing and colony identification by MALDI-TOF mass spectrometry. Results . Antibiotic-induced colonic dysbiosis in mice manifested itself as a decrease in the dominant microbiota and an increase in opportunistic pathogens. After 7 days of metabolite administration, the Lactobacillus population returned to normal in all treatment groups. The mice that received B. subtilis 3H metabolites showed the best results: their Lactobacillus spp. composition corresponded to that of intact animals. The content of Lac+ Escherichia coli returned to 100% in all treatment groups. After 21 days of metabolite administration, the authors observed the elimination of bacteria ( Rodentibacter spp., Aerococcus spp.) and fungi ( Trichosporon spp., Kazachstania spp.) in the B. subtilis 3H group; Trichosporon spp. (no effect on Kazachstania spp.) in the B. subtilis 1719 group; and Enterococcus spp., Kazachstania spp., and Trichosporon spp. (no effect on Rodentibacter spp. and Aerococcus spp.) in the commercial metabiotic group. Conclusions . Metabolites of B. subtilis strains 3H and 1719 help to restore the diversity and abundance of colonic microbiota in antibiotic-induced dysbiosis. The differences observed in microbiota re-establishment in the treatment groups indicate that there is interstrain variability in the probiotic activity of B. subtilis metabolites.
科学相关性。纠正生态失调的一个有希望的选择是使用代谢物,基于益生菌微生物的代谢物的产品。在发酵过程中,枯草芽孢杆菌(菌株3H和1719)在体外产生具有益生菌特性的代谢物。这些体外观察结果激发了在体内研究枯草芽孢杆菌代谢物对实验诱导的生态失调小鼠结肠粘膜微生物群的影响,并评估枯草芽孢杆菌代谢物作为代谢物的潜力。的目标。作者旨在比较枯草芽孢杆菌3H和枯草芽孢杆菌1719代谢物和商业代谢物在抗生素诱导的小鼠生态失调中的益生菌活性。材料和方法。作者通过腹腔注射庆大霉素诱导体重18-20 g的BALB/c小鼠实验性生态失调。为了进行后续校正,试验组给予吸收剂结合枯草芽孢杆菌代谢物,对照组给予含有枯草芽孢杆菌代谢物的商业代谢物(VKPM B-2335(3)3)灌胃注射,持续21天。结肠粘膜微生物群的定量和定性分析包括微生物培养和MALDI-TOF质谱鉴定。结果。抗生素诱导的小鼠结肠生态失调表现为优势菌群的减少和机会致病菌的增加。代谢物给药7 d后,各处理组乳酸杆菌数量均恢复正常。接受枯草芽孢杆菌3H代谢物的小鼠表现出最好的结果:它们的乳酸杆菌组成与未受影响的动物相当。各处理组Lac+大肠杆菌含量均恢复到100%。代谢产物给药21天后,作者观察到枯草芽孢杆菌3H组细菌(啮齿菌、气球菌)和真菌(Trichosporon、Kazachstania)的消除;枯草芽孢杆菌1719组中trichosporonspp(对Kazachstania spp无影响);商业代谢组对肠球菌、哈萨克斯坦球菌和毛磷菌(对啮齿杆菌和航空球菌没有影响)的抑制作用。结论。枯草芽孢杆菌菌株3H和1719的代谢物有助于在抗生素诱导的生态失调中恢复结肠微生物群的多样性和丰度。在处理组中观察到的微生物群重建的差异表明,枯草芽孢杆菌代谢物的益生菌活性存在菌株间变异。
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引用次数: 0
Determination of optimum nanofiltration conditions for the manufacturing process of human normal immunoglobulin G for intravenous administration 静脉注射用人正常免疫球蛋白G制备工艺最佳纳滤条件的确定
Pub Date : 2023-09-15 DOI: 10.30895/2221-996x-2023-23-3-1-400-410
N. V. Zubkova, A. M. Nikolaeva, A. V. Ivanov, O. V. Beliakova, M. V. Razumikhin, N. V. Vinokurova, I. S. Efimova, T. I. Smolyanova, E. I. Sakanyan
Scientific relevance . Medicinal products based on immunoglobulin class G (IgG) from human plasma are widely used in clinical practice to treat bacterial and viral infections, primary and secondary immunodeficiencies, and autoimmune diseases. Nanofiltration is a way to mitigate the risk of in-process contamination of raw materials with various pathogens, including viruses. Therefore, it is relevant to investigate the development and implementation of additional viral inactivation and/or elimination steps. Aim . This study aimed to develop and validate optimum nanofiltration conditions and to scale up the nanofiltration step for the manufacturing of human IgG for intravenous administration. Materials and methods . The study used a solution of IgG from plasma fractions II and III. The authors paired nanofilters manufactured by Planova 20N and BioEx (Asahi Kasei, Japan), Viresolve Pro (Merck Millipore, USA), Virosart HC and HF (Sartorius, Germany), and Pegasus SV4 and Prime (Pall, USA) with Sartopore polyethersulphone prefilters by Sartorius (Germany), Virosart MAX polyamide prefilters by Sartorius (Germany), and EKX-P regenerated cellulose prefilters by Pall (Germany). Virus reduction validation studies were performed with model viruses (human immunodeficiency virus type 1, porcine transmissible gastroenteritis virus, porcine parvovirus, murine encephalomyocarditis virus, and bovine viral diarrhoea virus) in the laboratories of the N.F. Gamaleya centre. The sample data analysis involved calculating mean values with 95% confidence intervals. Results . For all the selected combinations of prefilters and filters, the maximum nanofiltration throughput depended on the IgG concentration in the test solution. With the combination of an EKX-P filter with a Pegasus SV4 nanofilter, the maximum throughput and the IgG yield reached 6300 g/m 2 and 95%, respectively. When combined with a Planova 20N nanofilter, EKX-P and Sartopore (polyethersulphone) filters provided a maximum throughput of up to 2980 g/m 2 and an IgG yield of almost 100%, provided that the test solution had an IgG concentration of 10 g/L. With different filter combinations, virus reduction levels ranged from 4.00±0.05 to 4.75±0.04 log 10 for human immunodeficiency virus type 1, from 4.30±0.04 to 4.55±0.06 log 10 for porcine transmissible gastroenteritis virus, from 5.38±0.08 log10 to 5.57±0.04 log 10 for murine encephalomyocarditis virus, 5.12±0.10 log 10 to 5.25±0.08 log 10 for porcine parvovirus, and exceeded 5.00 log 10 for bovine viral diarrhoea virus. The virus reduction levels achieved were not statistically associated with prefilter brands. Conclusions . The study demonstrated that nanofiltration was effective at removing viruses with various virion sizes and physicochemical characteristics, including viruses as small as parvovirus B19. The levels of virus reduction exceeded 4 log 10 and met the acceptance criteria. The laboratory-scale nanofiltration parameters and the corresponding fil
科学相关性。以人血浆免疫球蛋白G (IgG)为基础的医药产品广泛用于临床治疗细菌和病毒感染、原发性和继发性免疫缺陷以及自身免疫性疾病。纳滤是一种减轻过程中各种病原体(包括病毒)污染原材料风险的方法。因此,研究其他病毒灭活和/或消除步骤的发展和实施是相关的。的目标。本研究旨在开发和验证最佳纳滤条件,并扩大纳滤步骤,用于制造静脉注射给药的人IgG。材料和方法。本研究使用血浆II和III部分的IgG溶液。作者将Planova 20N和BioEx (Asahi Kasei,日本)、Viresolve Pro (Merck Millipore,美国)、Virosart HC和HF (Sartorius,德国)、Pegasus SV4和Prime (Pall,美国)生产的纳米过滤器与Sartorius(德国)的Sartopore聚醚砜预过滤器、Sartorius(德国)的Virosart MAX聚酰胺预过滤器和Pall(德国)的EKX-P再生纤维素预过滤器配对。在N.F. Gamaleya中心的实验室中,用模型病毒(人类免疫缺陷病毒1型、猪传染性胃肠炎病毒、猪细小病毒、小鼠脑心肌炎病毒和牛病毒性腹泻病毒)进行了病毒还原验证研究。样本数据分析包括计算95%置信区间的平均值。结果。对于所有选择的预过滤器和过滤器组合,最大纳滤通量取决于测试溶液中的IgG浓度。当EKX-P过滤器与Pegasus SV4纳米过滤器组合使用时,IgG的最大通量和产率分别达到6300 g/ m2和95%。当与Planova 20N纳米过滤器结合使用时,EKX-P和Sartopore(聚醚砜)过滤器提供了高达2980 g/ m2的最大吞吐量和几乎100%的IgG产率,前提是测试溶液的IgG浓度为10 g/L。不同滤料组合下,人类免疫缺陷病毒1型的病毒减毒量为4.00±0.05 ~ 4.75±0.04 log10,猪传染性胃肠炎病毒减毒量为4.30±0.04 ~ 4.55±0.06 log10,鼠脑心肌炎病毒减毒量为5.38±0.08 ~ 5.57±0.04 log10,猪细小病毒减毒量为5.12±0.10 ~ 5.25±0.08 log10,牛病毒性腹泻病毒减毒量均超过5.00 log10。所达到的病毒减少水平与预过滤器品牌没有统计学关联。结论。研究表明,纳滤可以有效去除各种病毒粒子大小和物理化学特性的病毒,包括小到细小病毒B19的病毒。病毒减少程度超过4 log 10,并且符合可接受标准。实验规模的纳滤参数和相应的过滤次数,以及IgG的产率在放大过程中没有变化。因此,纳滤是一种有效的、高产的技术,有助于消除各种类型的病毒,并在不影响生物医药产品质量的情况下大大提高病毒的安全性。
{"title":"Determination of optimum nanofiltration conditions for the manufacturing process of human normal immunoglobulin G for intravenous administration","authors":"N. V. Zubkova, A. M. Nikolaeva, A. V. Ivanov, O. V. Beliakova, M. V. Razumikhin, N. V. Vinokurova, I. S. Efimova, T. I. Smolyanova, E. I. Sakanyan","doi":"10.30895/2221-996x-2023-23-3-1-400-410","DOIUrl":"https://doi.org/10.30895/2221-996x-2023-23-3-1-400-410","url":null,"abstract":"Scientific relevance . Medicinal products based on immunoglobulin class G (IgG) from human plasma are widely used in clinical practice to treat bacterial and viral infections, primary and secondary immunodeficiencies, and autoimmune diseases. Nanofiltration is a way to mitigate the risk of in-process contamination of raw materials with various pathogens, including viruses. Therefore, it is relevant to investigate the development and implementation of additional viral inactivation and/or elimination steps. Aim . This study aimed to develop and validate optimum nanofiltration conditions and to scale up the nanofiltration step for the manufacturing of human IgG for intravenous administration. Materials and methods . The study used a solution of IgG from plasma fractions II and III. The authors paired nanofilters manufactured by Planova 20N and BioEx (Asahi Kasei, Japan), Viresolve Pro (Merck Millipore, USA), Virosart HC and HF (Sartorius, Germany), and Pegasus SV4 and Prime (Pall, USA) with Sartopore polyethersulphone prefilters by Sartorius (Germany), Virosart MAX polyamide prefilters by Sartorius (Germany), and EKX-P regenerated cellulose prefilters by Pall (Germany). Virus reduction validation studies were performed with model viruses (human immunodeficiency virus type 1, porcine transmissible gastroenteritis virus, porcine parvovirus, murine encephalomyocarditis virus, and bovine viral diarrhoea virus) in the laboratories of the N.F. Gamaleya centre. The sample data analysis involved calculating mean values with 95% confidence intervals. Results . For all the selected combinations of prefilters and filters, the maximum nanofiltration throughput depended on the IgG concentration in the test solution. With the combination of an EKX-P filter with a Pegasus SV4 nanofilter, the maximum throughput and the IgG yield reached 6300 g/m 2 and 95%, respectively. When combined with a Planova 20N nanofilter, EKX-P and Sartopore (polyethersulphone) filters provided a maximum throughput of up to 2980 g/m 2 and an IgG yield of almost 100%, provided that the test solution had an IgG concentration of 10 g/L. With different filter combinations, virus reduction levels ranged from 4.00±0.05 to 4.75±0.04 log 10 for human immunodeficiency virus type 1, from 4.30±0.04 to 4.55±0.06 log 10 for porcine transmissible gastroenteritis virus, from 5.38±0.08 log10 to 5.57±0.04 log 10 for murine encephalomyocarditis virus, 5.12±0.10 log 10 to 5.25±0.08 log 10 for porcine parvovirus, and exceeded 5.00 log 10 for bovine viral diarrhoea virus. The virus reduction levels achieved were not statistically associated with prefilter brands. Conclusions . The study demonstrated that nanofiltration was effective at removing viruses with various virion sizes and physicochemical characteristics, including viruses as small as parvovirus B19. The levels of virus reduction exceeded 4 log 10 and met the acceptance criteria. The laboratory-scale nanofiltration parameters and the corresponding fil","PeriodicalId":32767,"journal":{"name":"Biopreparaty Profilaktika diagnostika lechenie","volume":"14 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135487068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Comparative analysis of quality attributes of a human albumin preparation with a modified stabilising composition 一种改良稳定成分的人白蛋白制剂质量特性的比较分析
Pub Date : 2023-09-13 DOI: 10.30895/2221-996x-2023-23-3-1-411-421
M. V. Tomilin, T. V. Korotkova, P. A. Loginov
Scientific relevance . The national and international human albumin preparations registered in the Russian Federation mainly differ in their excipient compositions. While all the international preparations of human albumin contain a mixture of sodium caprylate and N-acetyl-DL-tryptophan, the Russian ones contain only sodium caprylate. However, albumin stabilisation with sodium caprylate at high concentrations affects the ligand-binding properties of albumin. For this reason, as well as to achieve storage stability not only at temperatures of 2 °C to 8 °C but also at room temperature, most international manufacturers have reduced the sodium caprylate content in albumin preparations and added N-acetyl-DL-tryptophan. This demonstrates the relevance of studying the quality of a new Russian human albumin preparation with a modified stabilising composition, including both sodium caprylate and N-acetyl-DL-tryptophan. Aim . The study aimed at comparing several quality attributes of the human albumin preparation with a modified stabilising composition with those of imported human albumin preparations. Materials and methods . The human albumin preparation with a modified stabilising composition was manufactured by fractionation from donor plasma meeting the requirements of monograph FS.3.3.2.0001.19 of the State Pharmacopoeia of the Russian Federation edition XIV. The quality control was in line with the monograph on human albumin (FS.3.3.2.0006.18), and statistical analysis was conducted in Microsoft Excel in accordance with the general chapter on statistical analysis (OFS.1.1.0013.15). Results . The study preparation complied with the requirements specified in monograph FS.3.3.2.0006.18. All the manufactured batches were clear, thermostable, sterile, and non-pyrogenic. The prekallikrein activator levels were low (below 1 IU/mL). The aluminium content varied from 30.36±10.39 µg/L to 50.22±6.94 µg/L. The study preparation contained sodium ions at a concentration from 127.44±10.46 mmol/L to 145.59±7.32 mmol/L and less than 0.01 mmol/g of potassium ions. The osmolarity exceeded 240 mOsm/L. The content of α- and β-globulins ranged from 1.79±0.06% to 2.24±0.20%. The study preparation had a pH level of 6.9 to 7.2. The concentrations of polymers and aggregates did not exceed 0.5%. Conclusions . The quality attributes studied suggest that the human albumin preparation with a modified stabilising composition is comparable to its international counterparts and that it meets Russian and European pharmacopoeial standards.
科学相关性。在俄罗斯联邦注册的国家和国际人白蛋白制剂主要在赋形剂成分上有所不同。国际上所有的人白蛋白制剂都含有辛酸钠和n -乙酰- dl -色氨酸的混合物,而俄罗斯的制剂只含有辛酸钠。然而,高浓度辛酸钠稳定白蛋白会影响白蛋白的配体结合特性。因此,为了在2℃~ 8℃的温度下以及在室温下保持储存稳定性,国际上大多数厂家都降低了白蛋白制剂中辛酸钠的含量,并添加了n -乙酰基- dl -色氨酸。这证明了研究一种新的俄罗斯人白蛋白制剂的质量与改进的稳定成分的相关性,包括辛酸钠和n -乙酰基- dl -色氨酸。的目标。本研究旨在比较具有改良稳定成分的人白蛋白制剂与进口人白蛋白制剂的若干质量属性。材料和方法。采用从供体血浆中分离制备具有改良稳定成分的人白蛋白制剂,符合俄罗斯联邦国家药典第十四版专著FS.3.3.2.0001.19的要求。质量控制按照人白蛋白专著(FS.3.3.2.0006.18),按照统计分析通论(OFS.1.1.0013.15)在Microsoft Excel中进行统计分析。结果。本研究的准备工作符合FS.3.3.2.0006.18专著的要求。所有生产的批次都是透明的,耐热的,无菌的,无热原的。前钾likrein激活剂水平低(低于1 IU/mL)。铝含量变化范围为30.36±10.39µg/L ~ 50.22±6.94µg/L。钠离子浓度为127.44±10.46 mmol/L ~ 145.59±7.32 mmol/L,钾离子浓度小于0.01 mmol/g。渗透压超过240mosm /L。α-和β-球蛋白含量为1.79±0.06% ~ 2.24±0.20%。研究制剂的pH值为6.9至7.2。聚合物和聚集体的浓度不超过0.5%。结论。所研究的质量属性表明,具有改良稳定成分的人白蛋白制剂与国际同类产品相当,并且符合俄罗斯和欧洲药典标准。
{"title":"Comparative analysis of quality attributes of a human albumin preparation with a modified stabilising composition","authors":"M. V. Tomilin, T. V. Korotkova, P. A. Loginov","doi":"10.30895/2221-996x-2023-23-3-1-411-421","DOIUrl":"https://doi.org/10.30895/2221-996x-2023-23-3-1-411-421","url":null,"abstract":"Scientific relevance . The national and international human albumin preparations registered in the Russian Federation mainly differ in their excipient compositions. While all the international preparations of human albumin contain a mixture of sodium caprylate and N-acetyl-DL-tryptophan, the Russian ones contain only sodium caprylate. However, albumin stabilisation with sodium caprylate at high concentrations affects the ligand-binding properties of albumin. For this reason, as well as to achieve storage stability not only at temperatures of 2 °C to 8 °C but also at room temperature, most international manufacturers have reduced the sodium caprylate content in albumin preparations and added N-acetyl-DL-tryptophan. This demonstrates the relevance of studying the quality of a new Russian human albumin preparation with a modified stabilising composition, including both sodium caprylate and N-acetyl-DL-tryptophan. Aim . The study aimed at comparing several quality attributes of the human albumin preparation with a modified stabilising composition with those of imported human albumin preparations. Materials and methods . The human albumin preparation with a modified stabilising composition was manufactured by fractionation from donor plasma meeting the requirements of monograph FS.3.3.2.0001.19 of the State Pharmacopoeia of the Russian Federation edition XIV. The quality control was in line with the monograph on human albumin (FS.3.3.2.0006.18), and statistical analysis was conducted in Microsoft Excel in accordance with the general chapter on statistical analysis (OFS.1.1.0013.15). Results . The study preparation complied with the requirements specified in monograph FS.3.3.2.0006.18. All the manufactured batches were clear, thermostable, sterile, and non-pyrogenic. The prekallikrein activator levels were low (below 1 IU/mL). The aluminium content varied from 30.36±10.39 µg/L to 50.22±6.94 µg/L. The study preparation contained sodium ions at a concentration from 127.44±10.46 mmol/L to 145.59±7.32 mmol/L and less than 0.01 mmol/g of potassium ions. The osmolarity exceeded 240 mOsm/L. The content of α- and β-globulins ranged from 1.79±0.06% to 2.24±0.20%. The study preparation had a pH level of 6.9 to 7.2. The concentrations of polymers and aggregates did not exceed 0.5%. Conclusions . The quality attributes studied suggest that the human albumin preparation with a modified stabilising composition is comparable to its international counterparts and that it meets Russian and European pharmacopoeial standards.","PeriodicalId":32767,"journal":{"name":"Biopreparaty Profilaktika diagnostika lechenie","volume":"49 2 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135786750","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Development, certification, and use of a pharmacopoeial standard for the content of immunoglobulin A in human immunoglobulins for parenteral administration 制定、认证和使用用于肠外给药的人免疫球蛋白中免疫球蛋白a含量药典标准
Pub Date : 2023-09-13 DOI: 10.30895/2221-996x-2023-23-3-1-443-451
A. V. Nechaev, E. Yu. Kudasheva, E. L. Postnova, R. A. Volkova, O. V. Fadeikina, I. V. Borisevich, A. A. Movsesyants
Scientific relevance . The immunoglobulin A (IgA) impurity content in parenteral human immunoglobulins should be determined in accordance with the State Pharmacopoeia of the Russian Federation by kinetic nephelometry, radial immunodiffusion, or enzyme immunoassay (ELISA) with a reference standard. The International Standard (IS) for the content of IgA is certified using gravimetry and radial immunodiffusion. However, neither of the existing standards for the content of IgA in human immunoglobulins is currently certified using all three compendial methods. This prevents analysts from comparing test results obtained by different methods and may lead to an underestimation of the IgA content in human immunoglobulins. Aim . This study aimed to determine the procedure for the development, certification, and use of a pharmacopoeial reference standard (RS) for the content of IgA in human immunoglobulins. Materials and methods . The authors studied candidate RSs for the IgA content derived from human plasma for fractionation. The IgA content determination involved kinetic nephelometry, radial immunodiffusion, and ELISA, as well as commercial test kits and the IS. The authors quantified the IgA impurity in samples of commercial human immunoglobulins from various manufacturers. The data analysis involved descriptive statistics and variance analysis using Microsoft Excel and Statistica 10. Results . The authors established a pharmacopoeial standard with a certified IgA content of 1.98 mg/mL (expanded uncertainty, 0.44 mg/mL; coverage coefficient, k =2; confidence level, 95%) for IgA impurity quantification in human immunoglobulins by radial immunodiffusion and ELISA and that of 1.31–2.64 mg/mL (expanded uncertainty, 0.67 mg/mL; coverage ratio, k =3; confidence level, 99%) for intralaboratory quality control of IgA impurity quantification by kinetic nephelometry, radial immunodiffusion, and ELISA. Conclusions . The pharmacopoeial standard developed in the study has been included in the register of standards of the State Pharmacopoeia of the Russian Federation as the Reference Standard for the Content of Immunoglobulin Class A (IgA) (Registry No. 3.1.00454). The pharmacopoeial standard is intended for the standardisation of analytical methods for the-determination of the IgA impurity content in parenteral human immunoglobulins.
科学相关性。根据俄罗斯联邦国家药典的规定,采用动力学浊度法、径向免疫扩散法或酶免疫分析法(ELISA),结合参考标准品,测定肠外人免疫球蛋白A (IgA)杂质含量。IgA含量的国际标准(IS)采用重量法和径向免疫扩散法进行认证。然而,现有的人类免疫球蛋白中IgA含量标准目前都没有使用所有三种药典方法进行认证。这阻止了分析人员比较不同方法获得的测试结果,并可能导致低估人免疫球蛋白中的IgA含量。的目标。本研究旨在确定人免疫球蛋白中IgA含量的药典参考标准(RS)的开发、认证和使用程序。材料和方法。作者研究了分离人血浆中IgA含量的候选RSs。IgA含量的测定包括动力学浊度法、径向免疫扩散法和ELISA,以及商业检测试剂盒和IS。作者定量了来自不同厂家的商业人免疫球蛋白样品中的IgA杂质。数据分析包括描述性统计和方差分析,使用Microsoft Excel和Statistica 10。结果。建立药典标准,经认证IgA含量为1.98 mg/mL(扩展不确定度为0.44 mg/mL;覆盖系数,k =2;放射免疫扩散法和ELISA法测定人免疫球蛋白中IgA杂质含量的误差范围为1.31 ~ 2.64 mg/mL(扩展不确定度为0.67 mg/mL;覆盖率,k =3;置信水平为99%),用于动力学浊度法、径向免疫扩散法和ELISA法测定IgA杂质的实验室质量控制。结论。本研究制定的药典标准已作为A类免疫球蛋白(IgA)含量参考标准(注册号3.1.00454)列入俄罗斯联邦国家药典标准注册册。本药典标准旨在对人免疫球蛋白中IgA杂质含量测定的分析方法进行标准化。
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引用次数: 0
Effects of pertussis toxin and <i>Bordetella pertussis</i> lipo-oligosaccharide on the specific toxicity and potency of whole-cell pertussis vaccines 百日咳毒素与百日咳博德氏菌的作用&lt;/i&gt;脂寡糖对全细胞百日咳疫苗特异性毒性和效力的影响
Pub Date : 2023-09-13 DOI: 10.30895/2221-996x-2023-23-3-333-347
I. A. Alekseeva, I. V. Ibragimkhalilova, D. N. Lepekhova
Scientific relevance. The content of Bordetella pertussis lipo-oligosaccharide (LOS) and the residual levels of active pertussis toxin (PT) are generally accepted to be the primary factors that determine the reactogenicity of whole-cell pertussis vaccines. To improve the quality of whole-cell pertussis vaccines, it is both relevant and necessary to study the relationship between the toxicity of B . pertussis bacterial cell components and the main quality parameters of these vaccines, including their potency and specific toxicity, as termed in the WHO recommendations and the European Pharmacopoeia. Aim . This study aimed to analyse the effects of B . pertussis LOS and residual active PT on the specific toxicity and potency of adsorbed diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccines. Materials and methods . The authors tested 57 commercial batches of adsorbed DTwP vaccines for compliance with the regulatory standards and product specification files. Vaccine batches that failed specific toxicity tests formed Group 1, and the other batches were designated as Group 2. The potency was tested in F1 CBA/Ca×C57BL/6J hybrid mice with experimentally induced meningoencephalitis that were immunised with DTwP and reference vaccines. The authors assessed the specific toxicity of DTwP vaccines by changes in body weight following intraperitoneal administration. The toxic activity was assessed indirectly by changes in body weight in the first 16–24 h ( B . pertussis LOS) and on day 7 (PT) after dosing. The authors used Spearman’s rank correlation coefficient to measure the strength of correlation between the toxic activity of vaccine components and the specific toxicity and potency of the vaccine, which were established using the same vaccine batches. Results . The authors measured the toxic activity of LOS and residual active PT in the vaccine batches studied. The correlation coefficients between the specific toxicity and potency of the vaccines and the toxic activity of LOS were 0.113 ( p >0.05) and 0.049 ( p >0.05), respectively. Similarly, the correlation coefficients between the specific toxicity and potency of the vaccines and the toxic activity of PT accounted for 0.595 ( p <0.01) and –0.534 ( p <0.01), respectively. Conclusions . The authors studied the toxic activity of B. pertussis LOS and residual active PT in whole-cell pertussis vaccines and found an inverse correlation between the potency of the vaccines and the toxic activity of residual active PT. The study demonstrated that the specific toxicity test for whole-cell pertussis vaccines fails to detect and quantify B. pertussis LOS in the samples. The authors advise to determine the content of LOS in the B. pertussis strains intended for the production of whole-cell pertussis vaccines, which is not yet an accepted practice in the Russian Federation.
科学的相关性。百日咳杆菌脂寡糖(LOS)含量和活动性百日咳毒素(PT)残留水平被普遍认为是决定全细胞百日咳疫苗反应性的主要因素。为了提高全细胞百日咳疫苗的质量,研究B。百日咳细菌细胞成分和这些疫苗的主要质量参数,包括其效力和特定毒性,如世卫组织建议和欧洲药典所述。的目标。本研究旨在分析B。吸附白喉、破伤风和全细胞百日咳(DTwP)疫苗对百日咳LOS和残余活性PT的毒性和效力的影响材料和方法。作者测试了57个商业批次的吸附DTwP疫苗是否符合监管标准和产品规范文件。未通过特定毒性测试的疫苗批次形成第一组,其他批次被指定为第二组。用DTwP和参比疫苗免疫实验性脑膜脑炎F1 CBA/Ca×C57BL/6J杂交小鼠,检测其效力。作者通过腹腔注射后体重的变化评估了DTwP疫苗的特异性毒性。毒性活性通过前16 ~ 24 h的体重变化间接评估(B。百日咳LOS)和给药后第7天(PT)。作者使用Spearman等级相关系数来衡量疫苗成分的毒性活性与疫苗的特定毒性和效力之间的相关性强度,这是使用相同批次的疫苗建立的。结果。作者在研究的疫苗批次中测量了LOS和残余活性PT的毒性活性。疫苗的特异毒性和效力与LOS毒性活性的相关系数分别为0.113 (p >0.05)和0.049 (p >0.05)。同样,疫苗的特异毒性和效力与PT毒性活性的相关系数分别为0.595 (p <0.01)和-0.534 (p <0.01)。结论。作者研究了百日咳全细胞疫苗中百日咳B. LOS和残余活性PT的毒性活性,发现疫苗的效力与残余活性PT的毒性活性呈负相关。研究表明,全细胞百日咳疫苗的特异性毒性试验无法检测和定量样品中的百日咳B. LOS。作者建议确定用于生产全细胞百日咳疫苗的百日咳b型菌株中LOS的含量,这在俄罗斯联邦尚未被接受。
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引用次数: 0
Biopharmaceutical properties of polyvalent bacteriophage capsules 多价噬菌体胶囊的生物制药特性
Pub Date : 2023-09-13 DOI: 10.30895/2221-996x-2023-23-3-1-379-388
A. M. Nikolaeva, N. A. Kovyazina, E. V. Funkner, A. N. Krasilnikova, K. A. Lysko
Scientific relevance. Rational phage therapy is a highly effective way to combat bacterial infections, especially in conditions of steadily increasing antibiotic resistance. Most bacteriophage preparations are formulated as oral and topical solutions. However, oral administration of liquid phage preparations results in significant inactivation in the stomach. To shield active ingredients from the acidic environment, Sextaphag ® Pyobacteriophage, polyvalent, has been formulated into capsules. Aim . This study evaluated the polyvalent bacteriophage preparation in capsules in terms of its potency and biopharmaceutical properties. Materials and methods. The study compared the polyvalent bacteriophage preparation formulated as capsules with the polyvalent bacteriophage preparation formulated as a solution. The potency was evaluated by the Appelmans method, and phage particles were quantified by the Gratia method. To evaluate the acid-neutralising capacity, the authors placed test samples of the bacteriophage preparation in 0.1 M hydrochloric acid and analysed their potency by the Appelmans method. Chinchilla rabbits were used to analyse anti-phage immune responses, and their antibody levels were measured using an enzyme immunoassay test system developed by the authors. The pharmacokinetic parameters were studied in outbred white mice after oral dosing. Results. The polyvalent bacteriophage preparation exhibited high lytic activity towards Escherichia coli , Klebsiella pneumoniae , Proteus mirabilis , Proteus vulgaris , Pseudomonas aegidinosa , Staphylococcus aureus, and Streptococcus pneumoniae , which accounted for a dilution factor of 10 -6 . Following oral administration of the polyvalent bacteriophage preparation in capsules to mice, the level of absorption was 3.1–3.7 times higher than that observed with the solution. Repeated oral administration of therapeutic doses did not induce anti-phage antibodies in rabbits. The stability study showed that the polyvalent bacteriophage preparation retained high lytic activity for 18 months. Conclusions. According to the study results, the polyvalent bacteriophage preparation in capsules exerts significant antibacterial activity against the studied microorganisms, has a high level of absorption, retains its lytic activity for a long time, and does not induce anti-phage antibodies after oral dosing, which confirms its safety and efficacy.
科学的相关性。合理的噬菌体治疗是对抗细菌感染的一种非常有效的方法,特别是在抗生素耐药性稳步增加的情况下。大多数噬菌体制剂被配制成口服和局部溶液。然而,口服液体噬菌体制剂会导致胃中显著的失活。为了保护活性成分免受酸性环境的影响,Sextaphag®多价Pyobacteriophage已被配制成胶囊。的目标。本研究对多价噬菌体胶囊制剂的效力和生物制药性能进行了评价。材料和方法。本研究将多价噬菌体制剂配制成胶囊与多价噬菌体制剂配制成溶液进行了比较。用Appelmans法测定效价,用Gratia法测定噬菌体颗粒。为了评估噬菌体制剂的酸中和能力,作者将噬菌体制剂的测试样品置于0.1 M盐酸中,并通过Appelmans法分析其效力。用栗兔分析抗噬菌体免疫反应,并使用作者开发的酶免疫测定系统测量其抗体水平。口服给药后,对其在远交系小白鼠体内的药动学参数进行了研究。结果。该多价噬菌体制剂对大肠杆菌、肺炎克雷伯菌、奇异变形杆菌、寻常变形杆菌、盾绿假单胞菌、金黄色葡萄球菌和肺炎链球菌具有较高的裂解活性,其稀释倍数为10 -6。小鼠口服多价噬菌体胶囊制剂后,吸收水平比溶液高3.1-3.7倍。兔反复口服治疗剂量不诱导抗噬菌体抗体。稳定性研究表明,该多价噬菌体制剂在18个月内保持较高的裂解活性。结论。研究结果表明,多价噬菌体胶囊制剂对所研究的微生物具有显著的抗菌活性,吸收水平高,溶解活性保持时间长,口服给药后不诱导抗噬菌体抗体,证实了其安全性和有效性。
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引用次数: 0
Estimation of measurement uncertainty for the determination of loss on drying of biologicals 测定生物制品干燥损失的测量不确定度的估计
Pub Date : 2023-09-13 DOI: 10.30895/2221-996x-2023-23-3-1-452-462
O. V. Fadeikina, A. A. Voropaev, D. S. Davydov, R. A. Volkova
Scientific relevance . GOST ISO/IEC 17025-2019 requires testing laboratories to evaluate the measurement uncertainty of their results. Estimating the uncertainty of analytical methods intended for biologicals is a challenging task that requires time, effort, and a special approach. Measurement uncertainty estimation is of particular interest in the case of measuring loss on drying (LOD) for biologicals, since LOD testing procedures involve analysing measurements of a physical value, i.e. mass. Aim . This study aimed to estimate the measurement uncertainty of LOD determination in biological medicinal products. Materials and methods . The study examined a powdered active substance intended for a Bifidobacterium product (test sample). The authors conducted the LOD test in accordance with the State Pharmacopoeia of the Russian Federation (OFS.1.2.1.0010.15). Statistical processing of the results was performed using Microsoft Excel. To estimate the measurement uncertainty, the authors employed the bottom-up approach or used the standard deviation from testing results. Results . The authors identified the uncertainty components that affected the LOD determination results. When calculated using the bottom-up approach, the expanded uncertainty was 0.34% (coverage factor, k =2; approximate confidence level, 95%). In particular, the largest contributor to the expanded uncertainty was the uncertainty of measuring the mass of weighing bottles containing dried test samples (0.147%), whereas the smallest contributor was the uncertainty of weighing empty bottles (0.003%). When calculated using the standard deviation, the uncertainty of two parallel measurements amounted to 0.32%. Conclusions . Both approaches to calculating LOD measurement uncertainty yield comparable results. According to the uncertainty budget analysis, the uncertainty of measuring the mass of weighing bottles with dried test samples is the major contributor to the test result. For this reason, the conditions of sample preparation should be carefully controlled. The study results confirm that the LOD measurement uncertainty can be calculated using the standard deviation. Testing laboratory teams may benefit from the methods for identifying the factors influencing LOD test results and the methods for calculating the uncertainty of measurement described in this study.
科学相关性。GOST ISO/IEC 17025-2019要求测试实验室评估其结果的测量不确定度。估计用于生物制剂的分析方法的不确定度是一项具有挑战性的任务,需要时间、精力和特殊的方法。测量不确定度估计在测量生物制品干燥损失(LOD)的情况下特别有趣,因为LOD测试程序涉及分析物理值(即质量)的测量。的目标。本研究旨在对生物药品中LOD测定的测量不确定度进行估计。材料和方法。该研究检测了用于双歧杆菌产品(测试样品)的粉末状活性物质。作者按照俄罗斯联邦国家药典(OFS.1.2.1.0010.15)进行LOD试验。使用Microsoft Excel对结果进行统计处理。为了估计测量不确定度,作者采用了自底向上的方法或使用测试结果的标准偏差。结果。确定了影响LOD测定结果的不确定度成分。采用自底向上方法计算时,扩展不确定度为0.34%(覆盖因子,k =2;近似置信水平,95%)。特别是,扩展不确定度的最大贡献者是测量含有干燥测试样品的称重瓶的质量的不确定度(0.147%),而最小的贡献者是称重空瓶的不确定度(0.003%)。用标准偏差计算时,两次平行测量的不确定度为0.32%。结论。两种计算LOD测量不确定度的方法产生了可比较的结果。根据不确定度预算分析,用干燥试样测量称重瓶质量的不确定度是影响测试结果的主要因素。因此,应仔细控制样品制备的条件。研究结果证实了LOD测量不确定度可以用标准偏差来计算。测试实验室团队可以从本研究中描述的确定影响LOD测试结果的因素的方法和计算测量不确定度的方法中受益。
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引用次数: 0
On assessing the viral safety of individual units of the substance "Human plasma for fractionation" by nucleic acid amplification 核酸扩增技术评价“分离用人血浆”单个单位的病毒安全性
Pub Date : 2023-09-13 DOI: 10.30895/2221-996x-2023-23-3-1-463-473
E. V. Elbert, V. V. Nozhko, R. A. Volkova, A. A. Movsesyants, V. A. Merkulov, V. V. Kosenko
Scientific relevance . The absence of blood-borne viruses in human plasma-derived medicinal products must be ensured by the control of raw materials and the manufacturing process. Aim . This study aimed to analyse system suitability criteria for analytical procedures to assess the viral safety of individual units of the substance "Human plasma for fractionation" in terms of the content of nucleic acids of blood-borne viruses, considering the requirements of the European Pharmacopoeia. Materials and methods . The authors analysed individual units of the substance "Human plasma for fractionation" (hereinafter, plasma). The study used the International Standards (ISs) for human immunodeficiency virus RNA, hepatitis A virus (HAV) RNA, hepatitis C virus (HCV) RNA, hepatitis B virus (HBV) DNA, and parvovirus B19 DNA, as well as nucleic acid detection kits for these viruses based on polymerase chain reaction (PCR). Results . HCV RNA was not detected in any of the eight plasma samples studied, and parvovirus B19 DNA was detected in one of the samples at a concentration not exceeding 10 4 IU/mL. Three tests with the corresponding ISs showed that the studied reagent kits detected HCV RNA at a concentration of 10 2 IU/mL and parvovirus B19 DNA (M1 genotype) at a concentration of 10 4 IU/mL. In additional tests that were conducted in two samples considering the requirements of the European Pharmacopoeia for the detection of HCV RNA and parvovirus B19 DNA, a new batch of reagent kit I detected the HCV RNA IS at a concentration of 102 IU/mL only in one of three replicates, which did not correspond to the claimed sensitivity of the reagent kit. HCV RNA was not detected in either replicate in one of two plasma samples spiked with the HCV RNA IS at concentrations of 10 2 and 10 3 IU/mL, possibly because of plasma inhibitory properties. The sensitivity of the reagent kits to parvovirus B19 DNA corresponded to the label claims; the study did not show any inhibitory properties of the plasma samples. Conclusions . Polymerase chain reaction testing of the viral safety of plasma intended for manufacturing medicinal products should include control samples calibrated in IU/mL. Further research and appropriate pharmacopoeial reference materials are needed to set system suitability criteria for analytical procedures using such control samples.
科学相关性。必须通过对原料和生产过程的控制来确保人血浆来源的医药产品中不存在血源性病毒。的目标。本研究旨在考虑欧洲药典的要求,从血源性病毒核酸含量的角度,分析分析方法的系统适用性标准,以评估“分离用人血浆”物质的单个单位的病毒安全性。材料和方法。作者分析了单个单位的物质“人血浆分离”(以下简称血浆)。本研究使用了人类免疫缺陷病毒RNA、甲型肝炎病毒(HAV) RNA、丙型肝炎病毒(HCV) RNA、乙型肝炎病毒(HBV) DNA和细小病毒B19 DNA的国际标准(ISs),以及这些病毒基于聚合酶链反应(PCR)的核酸检测试剂盒。结果。8份血浆样本均未检测到HCV RNA,其中1份样本检测到细小病毒B19 DNA,浓度不超过104 IU/mL。3次检测结果表明,该试剂盒检测HCV RNA浓度为10 2 IU/mL,细小病毒B19 DNA (M1基因型)浓度为10 4 IU/mL。考虑到欧洲药典对HCV RNA和细小病毒B19 DNA检测的要求,在对两个样品进行的附加测试中,新一批试剂1仅在三个重复中的一个重复中检测到浓度为102 IU/mL的HCV RNA IS,这与试剂盒声称的灵敏度不符。HCV RNA在浓度为10.2和10.3 IU/mL的两个血浆样品中没有被检测到,可能是由于血浆抑制特性。试剂盒对细小病毒B19 DNA的敏感性符合说明书要求;该研究未显示血浆样品有任何抑制特性。结论。用于生产药品的血浆病毒安全性的聚合酶链反应检测应包括以IU/mL校准的对照样品。需要进一步的研究和适当的药典参考材料来为使用这些对照样品的分析方法制定系统适用性标准。
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引用次数: 0
Pollen allergen products: current standardisation issues 花粉过敏原产品:目前的标准化问题
Pub Date : 2023-09-13 DOI: 10.30895/2221-996x-2023-23-3-1-367-378
E. I. Sakanyan, M. A. Yasnaya, V. F. Vul, R. A. Bubenchikov, N. V. Vinokurova, E. S. Yurtaeva
Scientific relevance . Pollen allergen medicines are in high demand, and their therapeutic benefits directly correlate with their standardisation. Better diagnosis and treatment of allergic diseases require state-of-the-art procedures for assessing the allergenic activity of pollen allergen products using reference standards and physicochemical testing methods. Aim . The study aimed at developing methodological approaches to the standardisation of pollen allergen products in order to shift to measuring their potency in allergenic activity units (AAU) and bring their quality in line with the requirements of the State Pharmacopoeia of the Russian Federation. Materials and methods . The study used pollen allergen reference standards by Microgen, the WHO International Standard for timothy grass ( Phleum pratense ) pollen extract, a gel filtration standard kit of molecular weight markers ranging from 1.35 to 670 kDa, bovine serum albumin, serum samples with specific IgE obtained from donors sensitised to the study pollen allergens, labelled anti-human IgE antibodies, and reference standards for determining residual volatile solvents by gas chromatography. The identification of in-house reference standards for the potency of pollen allergens involved Western blotting (for allergenic components). The total protein content was determined by Bradford’s assay. In addition, the authors used high-performance liquid chromatography to study protein fractions and gas–liquid chromatography to determine the content of residual organic solvents. Results . To substitute the existing method of non-specific characterisation of allergenic activity in protein nitrogen units (PNU), the authors developed and tested a new method to control allergenic activity in allergenic activity units (AAU) based on an in vitro competitive enzyme-linked immunosorbent assay (ELISA) procedure developed and validated in this study. Furthermore, the authors developed and certified 15 primary in-house reference standards with allergenic activity established in AAU/mL using skin tests in vivo . The experimental data were analysed to standardise the allergenic activity of the pollen allergens manufactured by Microgen. The authors developed physicochemical methods for the certification of in-house reference standards and validated these methods in accordance with the State Pharmacopoeia of the Russian Federation. The study involved selecting chromatographic separation conditions for residual organic solvents (acetone and diethyl ether) and establishing system suitability criteria for the chromatographic system. The allergenic activity of secondary in-house reference standards was certified against that of primary in-house reference standards using competitive ELISA. Thus, the authors managed to shift to the standardisation of pollen allergen products in vitro . Conclusions . The authors developed their competitive ELISA-based method to standardise pollen allergen products by comparing the inhi
科学相关性。花粉过敏原药物的需求量很大,其治疗效果与其标准化直接相关。更好的诊断和治疗过敏性疾病需要使用参考标准和物理化学测试方法来评估花粉过敏原产品的致敏活性的最先进的程序。的目标。该研究旨在制定花粉过敏原产品标准化的方法学方法,以便转向以过敏原活性单位(AAU)测量其效力,并使其质量符合俄罗斯联邦国家药典的要求。材料和方法。本研究使用了Microgen的花粉过敏原参考标准、世卫组织蒂莫西草(Phleum pratense)花粉提取物国际标准、分子量为1.35至670 kDa的凝胶过滤标准试剂盒、牛血清白蛋白、从对研究花粉过敏原敏感的供体获得的具有特异性IgE的血清样本、标记的抗人IgE抗体,以及气相色谱法测定残余挥发性溶剂的参考标准。鉴定花粉过敏原效力的内部参考标准涉及Western blotting(致敏成分)。用Bradford法测定总蛋白含量。此外,采用高效液相色谱法研究蛋白质组分,气液色谱法测定残留有机溶剂的含量。结果。为了取代现有的蛋白氮单元(PNU)致敏活性的非特异性表征方法,作者开发并测试了一种基于体外竞争性酶联免疫吸附试验(ELISA)程序的控制致敏活性的新方法。此外,作者开发并认证了15个主要的内部参考标准,通过体内皮肤试验确定了AAU/mL的致敏活性。对实验数据进行分析,以规范Microgen公司生产的花粉过敏原的致敏活性。作者开发了内部参考标准认证的物理化学方法,并根据俄罗斯联邦国家药典对这些方法进行了验证。研究包括选择残留有机溶剂(丙酮和乙醚)的色谱分离条件和建立色谱系统的适宜性标准。二级内部标准品的致敏活性与一级内部标准品进行了竞争性ELISA验证。因此,作者设法将花粉过敏原产品的标准化转移到体外。结论。作者开发了基于elisa的竞争性方法,通过比较产品和标准对免疫反应的抑制来标准化花粉过敏原产品。该研究证明了用过敏原活性定量(在AAU中)代替蛋白氮含量测定(在PNU中)的可行性,并展示了使用AAU进行内部参考标准认证的第一个例子。此外,作者开发并验证了一种气液色谱法测定花粉过敏原产品中残留有机溶剂含量的分析方法。
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引用次数: 0
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Biopreparaty Profilaktika diagnostika lechenie
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