Endotoxin test by recombinant Factor C for 0.9% sodium chloride injection

Endotoxin contamination is a threat to the safety of pharmaceutical products, especially parenteral drugs. Any sterile and/or pyrogen-free pharmaceutical product requires regulatory specifications to ensure safe patient use. This study covers the performance evaluation study of an endotoxin quantitation commercial kit by recombinant Factor C (rFC), Endozyme II® Go, for 0.9% sodium chloride injection. The samples were spiked with endotoxin solutions between 0.0005 and 10 EU/mL and tested by the rFC kit to evaluate precision, accuracy, detection and quantification limits, linearity, and robustness. Each of the six points was assayed at least five times.The relative standard deviation for precision testing ranged from 1.9 to 8.3%. The recovery accuracy values of endotoxin were between 61% and 125% for the range from 0.005 to 10 EU/mL. The results demonstrated that the rFC method allows endotoxin quantification with accuracy, precision, specificity, and linearity for the range of 0.005 and 10 EU/mL for 0.9% sodium chloride injection. (AU)

A contaminação por endotoxinas é uma ameaça à segurança dos produtos farmacêuticos, especialmente dos medicamentos parenterais. Qualquer produto farmacêutico estéril e/ou livre de pirogênios requer especificações regulatórias para garantir a segurança de uso para o paciente. Este estudo abrange o estudo de avaliação de desempenho empregando o kit comercial Endozyme II® Go para quantificação de endotoxina, por Fator C recombinante (FCr), em amostras de cloreto de sódio 0,9% para uso parenteral. As amostras foram fortificadas com cinco concentrações distintas de soluções de endotoxina na faixa entre 0,0005 e 10 UE/mL. Cada um dos cinco níveis foi testado pelo menos cinco vezes para avaliação dos critérios de precisão, exatidão, limites de detecção e quantificação, linearidade e robustez. O desvio padrão relativo para os testes de precisão variou de 1,9 a 8,3%. Os valores de recuperação de endotoxina para o parâmetro exatidão estiveram compreendidos entre 61% e 125%. Os resultados demonstraram que o método por FCr permite a quantificação de endotoxinas com exatidão, precisão, especificidade e linearidade para a faixa de 0,005 e 10 UE/mL em amostras de cloreto de sódio 0,9% para uso parenteral. (AU)

Expression, purification, and functional verification of recombinant human glycoprotein hormone beta 5/alpha 2 fusion protein in CHO-S cells / 中国药理学通报

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Application value of bone cement containing rhbFGF and RHBMP-2 in PKP treatment of osteoporotic lumbar compression fracture / 中国骨伤

OBJECTIVE@#To investigate the effect of bone cement containing recombinant human basic fibroblast growth factor (rhbFGF) and recombinant human bone morphogenetic protein-2 (rhBMP-2) in percutaneous kyphoplasty(PKP)treatment of osteoporotic vertebral compression fracture(OVCF).@*METHODS@#A total of 103 OVCF patients who underwent PKP from January 2018 to January 2021 were retrospectively analyzed, including 40 males and 63 females, aged from 61 to 78 years old with an average of (65.72±3.29) years old. The injury mechanism included slipping 33 patients, falling 42 patients, and lifting injury 28 patients. The patients were divided into three groups according to the filling of bone cement. Calcium phosphate consisted of 34 patients, aged(65.1±3.3) years old, 14 males and 20 females, who were filled with calcium phosphate bone cement. rhBMP-2 consisted of 34 patients, aged (64.8±3.2) years old, 12 males and 22 females, who were filled with bone cement containing rhBMP-2. And rhbFGF+rhBMP-2 consisted of 35 patients, aged (65.1±3.6) years old, 14 males and 21 females, who were filled with bone cement containing rhbFGF and rhBMP-2. Oswestry disability index (ODI), bone mineral density, anterior edge loss height, anterior edge compression rate of injured vertebra, visual analog scale (VAS) of pain, and the incidence of refracture were compared between groups.@*RESULTS@#All patients were followed for 12 months. Postoperative ODI and VAS score of the three groups decreased (P<0.001), while bone mineral density increased (P<0.001), anterior edge loss height, anterior edge compression rate of injured vertebra decreased first and then slowly increased (P<0.001). ODI and VAS of group calcium phosphate after 1 months, 6 months, 12 months were lower than that of rhBMP-2 and group rhbFGF+rhBMP-2(P<0.05), bone mineral density after 6 months, 12 months was higher than that of rhBMP-2 and group calcium phosphate(P<0.05), and anterior edge loss height, anterior edge compression rate of injured vertebra of group rhbFGF+rhBMP-2 after 6 months and 12 months were lower than that of group rhBMP-2 and group calcium phosphate(P<0.05). There was no statistical difference in the incidence of re-fracture among the three groups (P>0.05).@*CONCLUSION@#Bone cement containing rhbFGF and rhBMP-2 could more effectively increase bone mineral density in patients with OVCF, obtain satisfactory clinical and radiological effects after operation, and significantly improve clinical symptoms.

High-level expression of anti FLAG tag antibody in plants / 生物工程学报

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.

Development and verification of a detection method for activity of neutralizing antibody in ELISA antibody positive serum of rats immunized with recombinant human interleukin-1 receptor antagonist / 中国生物制品学杂志

@#Objective To develop and verify a method for detecting the activity of neutralizing antibodies in ELISA antibody positive serum of rats immunized with recombinant human interleukin-1 receptor antagonist(rhIL-1Ra). Methods The SD rats were subcutaneously immunized with 3,20 and 100 mg/kg rhIL-1Ra injection respectively,10 rats in each group,half male and half female,twice a day at an interval of at least 4 h between each dose for 13 consecutive weeks. The blood samples were collected from the jugular vein of rats during the administration period and the recovery period. The serum samples were isolated and detected for the antibody titers by ELISA,and the samples positive for rhIL-1Ra antibody were purified by Protein A chromatographic column. Based on,D10G4·1 cells biological activity assay,a method for the detection of neutralizing antibody activity was developed and verified for the specificity,sensitivity and precision. The neutralizing antibody activity of rhIL-1Ra antibody positive serum determined by ELISA was detected by using the developed method.Results With the increase of doses,the serum antibody titers of rats in various dose groups gradually increased,and there were still antibodies in the recovery period,and the titer was still high. Rabbit anti-rhIL-1Ra monoclonal antibody showed obvious neutralizing effect on rIL-1Ra,while rabbit anti-rIFN-2b monoclonal antibody had no dose-effect relationship with rIL-1Ra. The sensitivity of the method was 171. 93 μg/mL;The CVs of precision verification were not more than 20%. The positive antibody sera detected by ELISA all had neutralizing effect on rhIL-1Ra injection,which was consistent with the results detected by ELISA. Conclusion The method developed in this study has good specificity and high sensitivity in the detection of serum neutralizing antibody activity in rats immunized with rhIL-1Ra,which can be used to detect the serum neutralizing antibody activity of animals with rhIL-1Ra repeated administration.

Establishment and validation of bioactivity measurement method for recombinant human midkine / 药学学报

To establish and optimize a method for the detection of recombinant human midkine (rhMK) activity and verify its methodology, cell counting kit-8 (cck-8) method was used to measure the proliferation activity of rat knee chondrocytes. The specificity, accuracy, precision, linearity and robustness of the method were also verified in this study. The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect; the recoveries of the samples with relative activities of 50%, 75%, 100%, 125%, 150% were in the range of 80.0% to 124.0% by statistical analysis, the relative standard deviations (RSD) of relative potency were all within 20%, the linear correlation coefficient, R2 ≥ 0.98, suggesting that the accuracy, precision and linearity of the method were good; the robustness correlation coefficient, R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5, indicating that robustness of the methods was good. In conclusion, a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development. This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center, Inc. (approval number: 2019-0008-06).

Effect of treatment with growth hormone on body composition and metabolic profile of short children born small for gestational age / Efeito do tratamento com hormônio de crescimento na composição corporal e no perfil metabólico de crianças nascidas pequenas para a idade gestacional com baixa estatura

ABSTRACT Objective: To assess the effect of recombinant growth hormone (rGH) on body composition and metabolic profile of prepubertal short children born small for gestational age (SGA) before and after 18 months of treatment. Methods: It is a clinical, non-randomized, and paired study. Children born SGA, with birth weight and/or length <-2 standard deviations (SD) for gestational age and sex, prepubertal, born at full term, of both genders, with the indication for treatment with rGH were included. The intervention was performed with biosynthetic rGH at doses ranging from 0.03 to 0.05 mg/kg/day, administered subcutaneously, once a day at bedtime. Total lean mass (LM) and total fat mass (FM) were carried out using dual-energy X-ray absorptiometry (DXA), and the metabolic profile was assessed for insulin, glycemia, IGF-1 levels and lipid profile. Results: Twelve patients (nine girls, 8.17±2.39 y) were evaluated; three patients dropped out of the study. There was an increase of LM adjusted for length (LMI) (p=0.008), LMI standard deviation score (SDS) adjusted for age and sex (p=0.007), and total LM (p<0.001). The percentage of body fat (BF%) and abdominal fat (AF) remained unaltered in relation to the beginning of treatment. Among the metabolic variables, blood glucose remained within normal levels, and there was a reduction in the number of participants with altered cholesterol (p=0.023). Conclusions: The effect of rGH treatment was higher on LM than in FM, with increased LM adjusted for length and standardized for age and sex. Glycemia remained within the normal limits, and there was a decreased number of children with total cholesterol above the recommended levels.

RESUMO Objetivo: Avaliar o efeito do hormônio de crescimento recombinante (rHC) na composição corporal e no perfil metabólico de crianças pré-púberes com baixa estatura, nascidas pequenas para a idade gestacional (PIG) antes e depois de 18 meses de tratamento. Métodos: Estudo clínico, não randomizado e pareado. Foram incluídas crianças nascidas PIG, com peso e/ou altura ao nascer <-2 desvios padrão (DP) para idade gestacional e sexo, pré-púberes, nascidas a termo, de ambos os sexos, com indicação de tratamento com rGH. A intervenção foi realizada com rGH biossintético com doses variando de 0,03 a 0,05 mg/kg/dia, administrado por via subcutânea, uma vez ao dia ao deitar-se. A massa magra total (LM) e a massa gorda total (MG) foram determinadas por meio de absorciometria de raios X de dupla energia (DXA), e o perfil metabólico foi avaliado com dosagens de insulina, glicemia, IGF-1 e perfil lipídico. Resultados: Doze pacientes (nove meninas, 8,17±2,39 anos) foram avaliados; três pacientes abandonaram o estudo. Houve aumento da LM ajustada para estatura (LMI) (p=0,008), LMI standard deviation scores (SDS) ajustada para idade e sexo (p=0,007) e LM total (p<0,001). O percentual de gordura corporal (GC%) e gordura abdominal (AF) permaneceu inalterado em relação ao início do tratamento. Entre as variáveis metabólicas, a glicemia manteve-se na normalidade, e houve redução do número de participantes com colesterol alterado (p=0,023). Conclusões: O efeito do tratamento com HCr foi maior na MM do que na MG, com o aumento da MM ajustada para altura e padronizada para idade e sexo. A glicemia permaneceu normal e houve redução do número de crianças com colesterol total acima do recomendado.

Obtención de la proteína verde fluorescente recombinante y su anticuerpo policlonal Igy

La Proteína Verde Fluorescente (Green Fluorescent Protein, GFP) es ampliamente utilizada en ensayos in vivo e in vitro. Se han generado múltiples variantes de esta proteína para diversificar sus características, como la GFP-enhancer (EGFP) que emite una señal de fluorescencia 35 veces mayor en comparación con la proteína silvestre, siendo implementada como proteína fusión en estudios de localización y estabilidad estructural, entre otros. La detección de esta proteína y sus variantes puede ser directa o indirecta, mediante el uso de anticuerpos anti-GFP. Aunque el uso de GFP es generalizado y de evidente utilidad en investigación y en docencia, los insumos para su estudio exhiben un alto costo dado que deben ser importados, constituyendo un recurso limitado en Colombia. El presente trabajo reporta la clonación y expresión de la proteína recombinante 6xHisEGFP, cuya purificación se completó a partir de la fracción soluble e insoluble del sistema heterólogo Escherichia coli mediante cromatografía de afinidad a metales inmovilizados y electroforesis preparativa, respectivamente. La proteína purificada se implementó como antígeno para la producción de anticuerpos policlonales aviares (IgY) contra la EGFP, los cuales se obtuvieron desde los huevos colectados y el suero de las sangrías de las gallinas inmunizadas. En este sentido, la estrategia metodológica planteada constituye un avance en el desarrollo de un sistema biotecnológico para la producción nacional de herramientas moleculares como los anticuerpos policlonales aviares a bajo costo.

Green Fluorescent Protein (GFP) is widely used in in vivo and in vitro assays. Multiple variants of this protein have been generated to diversify its characteristics, such as the enhancer GFP (EGFP) that emits a 35-fold higher fluorescence signal compared to the wild-type protein, being implemented as a fusion reporter in localization and structural stability studies, among others. Detection of this protein can be direct or indirect, fusing anti-GFP antibodies. Although the use of GFP is generalized and of evident utility in research and teaching, the molecular tools for its study exhibit a high cost since they must be imported, constituting a limited resource in Colombia. This work reports the cloning and expression of the recombinant protein 6xHisEGFP, which purification was completed from the soluble and insoluble fraction of the heterologous Escherichia coli system by immobilized metal affinity chromatography and preparative SDS-PAGE, respectively. The purified protein was implemented as an antigen to produce avian polyclonal antibodies (IgY) against EGFP, which were obtained from collected eggs and blood serum from immunized hens. In this sense, the proposed methodological strategy constitutes an advance in the development of a biotechnological system for the national production of molecular tools such as avian polyclonal antibodies at low-cost.

The Use of Escherichia coli for Recombinant Human Insulin Production

Insulin is the essential hormone produced by the pancreas. which is accountable for sanctioning glucose we acquire from our food sources to be deposited in our body cells. Without insulin, our bodies cannot control blood sugar levels, so insulin is a vital hormone for survival. A diabetic person either does not produce insulin or is resilient to it for a multiple reasons. Because of this, they need insulin injections to process glucose. It has become stress-free for patients around the world to acquire insulin with the production of recombinant human insulin produced by Escherichia coli . This short review will provide an overview of the steps engaged in constructing recombinant human insulin utilizing the K12 strain of E. coli along with the prominence of recombinant insulin and why E. coli is most commonly used for insulin production.

Angioedema after rt-PA infusion led to airway emergency: a case report of rescue treatment with fresh frozen plasma

Abstract The authors report the case of a 71-year-old woman presented to the Emergency Department with acute ischemic stroke. She was treated with rt-PA and interventional endovascular revascularization and developed rapidly progressing angioedema that led to emergency intubation. The standard treatment was not very effective and the swelling improved after infusion of fresh frozen plasma. Angioedema after rt-PA infusion could be a life-threatening emergency that requires quick airway management by skilled professionals. As this condition is triggered by several factors, such as unregulated histamine and bradykinin production, the traditional treatment recommended by the guidelines may not be sufficient and the use of FFP can be considered as a safe and valuable aid.

Osteometabolic profile and bone mass in the transition phase: ethnic differences in Brazilians treated with somatropin during childhood

Abstract Objective: The main growth hormone action is to promote linear growth increasing protein synthesis stimulation and osteoblastic activity. Peak bone mass extends from adolescence to 30 years of age. Several factors may influence this acquisition and prevent fracture risk in adulthood, such as genetic potential, GH, ethnicity, and lifestyle factors. This study aims to compare bone mass and osteometabolic profile of white and Afro-descendant Brazilian adolescents in the transition phase, who were treated with human recombinant growth hormone in childhood. Methods: The authors selected 38 adolescents from the Transition Outpatient Clinic of the University of São Paulo. Lumbar spine and total body bone mineral density (BMD) and bone mineral content (BMC), serum calcium, 25-OH-vitamin D and bone markers were analyzed at the rhGH withdrawal. Results: The mean age was 16.8 ± 1.6 years. There were 21 Afro-descendant and 17 whites. Thirty-four percent of the sample presented vitamin D insufficiency, 66% inadequate calcium intake and 44.7% physical inactivity. The Afro-descendants showed a lower lumbar spine and total body Z scores than those of the whites (p = 0.04 and p = 0.03, respectively), as well as their mean body weight (p = 0.03). There were no differences in the remaining osteometabolic parameters. Conclusion: As most adolescents had vitamin D insufficiency, low calcium intake, and physical inactivity, calcium, and cholecalciferol supplementation and lifestyle changes should be encouraged. The Brazilian Afro-descendant may be a vulnerable group for low bone mass, requiring

Successful outcomes of surgery procedures in patient with Glanzmann´s Thrombasthenia

We report a 35-year-old female patient with Glanzmann's thrombasthenia (GT) and severe anemia due to abnormal uterine bleeding secondary to uterine myomatosis. She required several admissions of red blood cells and platelet transfusions. An elective subtotal hysterectomy with salpingo-oophorectomy was proposed and recombinant factor VII was required. Surgical and postoperative outcomes were successful, without surgical complications, bleeding, or hemogram alterations. 4 years later, she required tooth extraction because of periodontal disease and pulp necrosis. In Peru, reports of GT patients requiring major and minor surgical procedures are lacking, given the low disease prevalence and the difficulties related to surgery. The report of these successful cases becomes relevant to continue improving GT management.

Presentamos el caso de una paciente de 35 años con trombastenia de Glanzmann (GT) y anemia severa por sangrado uterino anormal secundario a miomatosis uterina. Requirió varias admisiones de transfusiones de glóbulos rojos y plaquetas. Se propuso histerectomía subtotal electiva con salpingo-ooforectomía y se requirió factor VII recombinante. Los resultados quirúrgicos y postoperatorios fueron exitosos, sin complicaciones quirúrgicas, sangrado ni alteraciones del hemograma. 4 años después, requirió extracción dental por enfermedad periodontal y necrosis pulpar. En Perú faltan reportes de pacientes con GT que requieran procedimientos quirúrgicos mayores y menores, dada la baja prevalencia de la enfermedad y las dificultades relacionadas con la cirugía. El reporte de estos casos de éxito cobra relevancia para seguir mejorando la gestión de GT

Molecular characterization & recombination analysis of complete enterovirus-88 isolated from acute flaccid paralysis cases in India

Background & objectives: Focus on non-polio enteroviruses (NPEVs) causing acute flaccid paralysis (AFP) due to myelitis has increased with the containment of the poliovirus. Enterovirus-B88 (EV-B88) has been associated with the AFP cases in Bangladesh, Ghana, South Africa, Thailand and India. In India, EV-B88 infection was linked to AFP a decade ago; however, to date, no complete genome has been made available. In this study, the complete genome sequence of EV-B88 was identified and reported from two different States (Bihar and Uttar Pradesh) in India using the next-generation sequencing technique. Methods: Virus isolation was performed on the three AFP suspected cases as per the WHO-recommended protocol. Samples showing cytopathic effects in the human Rhabdocarcinoma were labelled as NPEVs. Next-generation sequencing was performed on these NPEVs to identify the aetiological agent. The contiguous sequences (contigs) generated were identified, and reference-based mapping was performed. Results: EV-B88 sequences retrieved in our study were found to be 83 per cent similar to the EV-B88 isolate from Bangladesh in 2001 (strain: BAN01-10398; Accession number: AY843306.1). Recombination analyses of these samples demonstrate recombination events with sequences from echovirus-18 and echovirus-30. Interpretation & conclusions: Recombination events in the EV-B serotypes are known, and this work reconfirms the same for EV-B88 isolates also. This study is a step in increasing the awareness about EV-B88 in India and emphasizes future studies to be conducted in the identification of other types of EV present in India.

Recombinant Human Peroxiredoxin-5 Improves Anti-tumor Immunity by Inducing Macrophage Polarization / 中国生物化学与分子生物学报

Tumor cells can use different strategies to suppress the immune system and disable them for killing tumor cells. Previous studies have shown that recombinant human peroxiredoxin-5 (hPRDX5) can activate the normal anti-tumor immune, so as to control and eliminate the tumor cells, but its exact mechanism of action needs to be studied in depth. The study aimed to investigate whether hPRDX5 exerts its anti-tumor activity by activating or reversing the polarization state of mouse macrophages RAW264. 7 cells. The results of CCK8 showed that different doses of hPRDX5 could significantly enhance the viability of macrophage compared with the control group (P < 0. 001); The results of Nitric oxide (NO) test showed that hPRDX5 significantly enhanced NO secretion levels in RAW264. 7 cells (P < 0. 001); ELISA experiments revealed that hPRDX5 promotes TNF-α (P<0. 01) and IL-6 (P<0. 001) secretion in RAW264. 7 cells; Flow cytometry revealed that hPRDX5 increased the expression of antigen differentiation cluster (CD) 80 (P < 0. 01) and inducible nitric oxide oxide synthase (iNOS) (P < 0. 001) in RAW264. 7 cells, and reduced the expression of CD206 (P < 0. 001) in RAW264. 7 cells induced by tumor conditional culture solution (TCS); Lactate dehydrogenase (LDH) experiments revealed that hPRDX5 can increase the killing activity of mouse macrophages on mouse pancreatic cancer Panc02 cells. hPRDX5 is able to activate mouse macrophage RAW264. 7 cells, promotes its M1-type polarization, reverses M2-type polarization, and exerts antitumor activity through the immune-enhancing effect.

Clinical efficacy of intracoronally targeted recombinant human urokinase combined with percutaneous coronary intervention (PCI) in acute myocardial infarction (AMI) with high thrombus burden / 中国临床药理学与治疗学

AIM: To study the effect of of intracoronally targeted recombinant human urokinase combined with percutaneous coronary intervention (PCI) in acute myocardial infarction (AMI) with high thrombus burden. METHODS: In this retrospective analysis study, 85 AMI patients with heavy thrombus burden admitted to Wuhu Second people's Hospital for percutaneous coronary intervention (PCI) from November 2020 to November 2022 were divided into observation group (n=37) and control group (n=48) according to different treatment methods.Recombinant human urokinase were used for coronary intervention in observation group. The control group was not treated with recombinant human urokinase. The myocardial injury markers troponinI (cTnI) and creatine kinase (CK) within 24 h after PCI, the percentage of segment resolution≥50% 1 h after PCI, intraoperative coronary lesions blood flow, the incidence of adverse cardiovascular events (MACE) during hospitalization, and cardiac function indexleft ventricular end diastolic (LVED), fractional shortening (FS), left ventricular ejection fraction (LVEF) level change one month discharge were compared between the two groups after PCI. RESULTS: After PCI, the levels value of cTnI and creatine kinase in the observation group at within 24 h after PCI were (69.35±16.31) ng/mL vs. (80.52±15.20) ng/mL, (3 136.27±1 952.52) U/L vs. (4 554.51±1 982.34) U/L, which were significantly lower than those in the control group (P0.05) CONCLUSION: Intracoronally targeted application of recombinant human urokinase combined with percutaneous coronary intervention (PCI) has a significant effect on AMI with heavy thrombus burden, which can effectively improve cardiac function, coronary blood flow and myocardial reperfusion, and reduce myocardial damage without increasing the risk of MACE

Expression, purification and functional verification of recombinant human α-galactosidase A in suspension CHO-S / 中国药理学通报

Aim To express and purify rhα-Gal A with a 6 X His tag via using a serum-free expression system in high-density suspension culture of Chinese hamster ovary cells ( CHO-S) , and to verify the scavenging effect of rhα-Gal A on globular trisaccharide ceramide (Gb3 or GL3) . Methods The construction of recombinant protein expression vector, pcDNA4-GLA, was achieved by fusing the human α-galactosidase cDNA, gla, with 6 X His tag and artificial DNA synthesis. The expression plasmid was transfected into the suspended CHO-S to express rhα-Gal A and then purified. Following this procedure, we determined rhα-Gal A's expression, the enzymatic activity, and the glycosylation of the recombinant enzyme. Co-incubation with cultured cells was performed to examine whether rhα-Gal A could be taken up into the cells and effectively remove Gb3 substrates. Results rhα-Gal A was successfully expressed and purified after transiently transfecting pcDNA4-GLA into the suspended CHO-S, and the yield was up to (100 ±20. 6) mg • L

An injectable signal-amplifying device elicits a specific immune response against malignant glioblastoma

Despite exciting achievements with some malignancies, immunotherapy for hypoimmunogenic cancers, especially glioblastoma (GBM), remains a formidable clinical challenge. Poor immunogenicity and deficient immune infiltrates are two major limitations to an effective cancer-specific immune response. Herein, we propose that an injectable signal-amplifying nanocomposite/hydrogel system consisting of granulocyte-macrophage colony-stimulating factor and imiquimod-loaded antigen-capturing nanoparticles can simultaneously amplify the chemotactic signal of antigen-presenting cells and the "danger" signal of GBM. We demonstrated the feasibility of this strategy in two scenarios of GBM. In the first scenario, we showed that this simultaneous amplification system, in conjunction with local chemotherapy, enhanced both the immunogenicity and immune infiltrates in a recurrent GBM model; thus, ultimately making a cold GBM hot and suppressing postoperative relapse. Encouraged by excellent efficacy, we further exploited this signal-amplifying system to improve the efficiency of vaccine lysate in the treatment of refractory multiple GBM, a disease with limited clinical treatment options. In general, this biomaterial-based immune signal amplification system represents a unique approach to restore GBM-specific immunity and may provide a beneficial preliminary treatment for other clinically refractory malignancies.

Clinical observation of herombopag combined with recombinant human thrombopoietin in the treatment of primary immune thrombocytopenia / 中国药房

OBJECTIVE To investigate the clinical efficacy and safety of herombopag combined with recombinant human thrombopoietin （rhTPO） in the treatment of primary immune thrombocytopenia （ITP） in the real world. METHODS A retrospective study was conducted on the patients diagnosed with ITP in the Second Affiliated Hospital of Bengbu Medical College from January 2021 to December 2022. Among them， 98 patients who were treated with a combination of herombopag and rhTPO were included in the observation group， and 157 patients who were treated with rhTPO alone were included in the control group. The changes in platelet count， clinical efficacy， bleeding， platelet transfusion rate and adverse drug reactions before and after treatment were observed and compared between the two groups. RESULTS Since the 8th day of treatment， there was a statistically significant difference in platelet count between the two groups （[ 61.04±13.46）×109 L－1 in observation group， （52.11±12.06）× 109 L－1 in control group] （P＜0.05）， and there also was a statistically significant difference in the peak and stable values of platelet count between the two groups （P＜0.05）. The total effective rates of the observation group and the control group were 79.59% and 66.88%， with cumulative response rates of 81.32% and 68.68%， and median response durations of 8 days and 10 days， respectively； these differences were statistically significant （P＜0.05）. During the treatment period， the bleeding rates of the observation group and control group were 3.06% and 8.28% （P＜0.05）， bleeding events were categorized as grade 1 or 2， and platelet transfusion rates were 31.63% and 40.76%； the differences in bleeding rates and platelet transfusion rates between the two groups was statistically significant （P＜0.05）. The incidences of adverse drug reactions in the two groups were 11.22% and 9.55%， respectively， with no statistically significant difference （P＞0.05）， and no moderate to severe adverse drug reaction was found. CONCLUSIONS The combination of herombopag and rhTPO can significantly increase platelet levels and response rate， and reduce bleeding rate and platelet transfusion rate in ITP patients， with good safety.

Optimization and verification of size exclusion chromatography-high performance liquid chromatography for determination of recombinant human growth hormone-Fc immunofusion protein polymer / 中国生物制品学杂志

@#Objective To optimize and verify the size exclusion chromatography-high performance liquid chromatography(SEC-HPLC) method for the determination of recombinant human growth hormone(rhGH)-Fc immunofusion protein polymer.Methods The multimer content of rhGH-Fc immunofusion protein was detected by SEC-HPLC.The detection conditions(salt concentration,mobile phase pH,flow rate,column temperature and column model) were optimized to observe the separation effect of the target proteins and polymers.The system suitability,specificity,linearity and range,precision,accuracy and limit of quantification of the method were verified.Results The optimized method was to use TSK-gel G2000SW_(x1)column(5 μm,7.8 mm × 300 mm),mobile phase of 50 mmol/L phosphate buffer(pH 6.80),detection wavelength of280 nm,injection volume of 100 μL,flow rate of 0.6 mL/min and column temperature of 45 ℃.The resolution of rhGHFc immunofusion protein and polymer,the theoretical plate number and the tailing factor all met the requirements;the peak time of rhGH-Fc immunofusion protein was the same as that of the control,while the peak time of GH national standard was different from that of the control,and the protein buffer showed no peak;the concentration of rhGH-Fc immunofusion protein was in the range of 0.307～1.842 mg/mL with good linear correlation between the peak area integral value and the injection volume(R~2=0.999 4);the RSD of peak area and purity in repeatability verification were 0.7% and 0.1%,respectively;the RSD of intermediate precision verification was 0.8%;the average recovery rate of accuracy verification was 99.1% with the RSD of 1.9%;the limit of quantification was 6 μg/mL.Conclusion The optimized SEC-HPLC method was used to detect the content of polymer in rhGH-Fc immunofusion protein with improved accuracy,and the column efficiency and separation were in accordance with the relevant requirements of Chinese Pharmacopoeia(Volume Ⅳ,2020edition),which could be used for the detection of polymer content in samples.

High level expression of recombinant human serum albumin in CHO cells and optimization of its culture technology / 中国生物制品学杂志

@#ObjectiveTo achieve efficient expression of human serum albumin(HSA)in Chinese hamster ovary(CHO)cells and optimize its culture technology,so as to lay a foundation of the large-scale production of HSA.MethodsThe eukaryotic expression vector of HSA was constructed by gene recombination technology,and then electrotransfected into fully suspended CHO cells. The monoclonal cell lines with stable and high expression of HSA were screened by G418 and limited dilution method. By adding glucose,sodium butyrate and supplementalmedium to the basal medium,the cell culture process was optimized to improve the expression of HSA. Finally,the scale-up culture verification was carried out in a 5 L bioreactor.ResultsThe recombinant expression vector pcDNA3.1-HSA was successfully constructed and expressed in fully suspended CHO cells. After two monoclonal screening,the secondary monoclonal cell lines CHO-rHSA-7H2A9 and CHOrHSA-7H2D12 were obtained with high HSA expression of 29. 37 mg/L and 25. 26 mg/L respectively. The HSA expression level reached about 100. 00 mg/L by optimizing the culture process and wasfinally increased to 166. 16 mg/L in the 5 L bioreactor,which was about 30 times higher than that in the supernatant of the first monoclonal cells.Conclusion The high level expression of HSA in CHO cells was achieved,which laid a foundation of the further large-scale production of HSA in the field of biological products and solving the market supply problems.