Quality control of widely used therapeutic recombinant proteins by a novel real-time PCR approach

Background: Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase [rSK], and alpha interferon [IFN-alpha] preparations

Methods: A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilu ons of DNA extracted fromE. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-alpha samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry

Results: The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-alpha preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA

Conclusion: Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers' minds on refining drugs, and provides consumers with safer biopharmaceuticals

Evaluation of cisplatin and cisplatin-loaded magnetic iron oxide nanoparticles on BCL2 and BAX genes in the breast cancer T47D cell line

Breast cancer is the second leading cause of cancer death in women. Cisplatin is a traditional cancer drug commonly used in chemotherapy for killing cancer cells. Modulation at the mRNA levels of apoptotic related genes often correlate with the sensitivity of various types of cancer cells to chemotherapeutic agents. Nanoparticulate drug delivery systems are being developed to effectively deliver smaller doses of chemotherapeutic agents and control drug distribution in the body. In this study, we evaluate the expressions of BCL2 and BAX genes in T47D treated with cisplatin and cisplatin nanoparticles, which can result in a new approach to breast cancer therapy. In this study, we treated T47D cells with different concentrations of cisplatin and cisplatin nanoparticles at 48 h. The IC50 was determined. We extracted RNA by using RNX solution, after which cDNA was synthesized. The precise primers for the BCL2, BAX and TBP genes were designed by specific software. The quantity of BCL2 and BAX gene expression compared to TBP gene [reference gene] was analyzed using real-time PCR. BCL2 and BAX gene expression levels in T47D cells treated by cisplatin were 0.7 [BCL2] and 1.48 [BAX], in T47D cells treated with cisplatin-loaded nanoparticles, the gene expressions were 0.03 [BCL2] and 2.41 [BAX]. In this study, the results have shown that cisplatin-loaded nanoparticles are effective anticancer agents. Cisplatin nanoparticles induce apoptosis in human breast cancer cell lines. We have shown that cisplatin nanoparticles strongly increased cytotoxicity in comparison to the free drug in the T47D cell line

[Development and application of real-time quantitative polymerase chain reaction technique using SYBR green I in the diagnosis of Down syndrome]

Rapid diagnosis of Trisomy 21 Syndrome [Down Syndrome] patients using Real-Time quantitative Polymerase Chain Reaction [Real-Time qPCR] in order to establish a novel method for prenatal diagnosis in the future. A total of 5 ml of peripheral blood was obtained from each patient and normal controls [NR]. Then, genomic DNA from lymphocytes was extracted using the salting out procedure. Gene dosage levels of DSCAM and [PMP22, DSCAM] in Down Syndrome and NR were analyzed using real-time quantitative PCR. The DSCAM/ PMP22 ratio was calculated according to the 2-delta delta Ct formula for all samples. Real-time PCR showed a DSCAM/PMP22 ratio of 1.48 +/- 0.18 and 1.01 +/- 0.10 [p<0.001] in Down Syndrome and normal samples, respectively, demonstrating three copies of the target [DSCAM] gene in Trisomy 21 Syndrome. DSCAM/PMP22 ratio is increased significantly in Down Syndrome patients than NR [1.5 times]. Therefore, the real-time quantitative PCR technique can be used as a sensitive, accurate and reliable technique for rapid and prenatal diagnosis of Trisomy 21 Syndrome

Use of Real-Time PCR method on the amniocyte samples for prenatal diagnosis of Down syndrome

In this study, the possibility of prenatal diagnosis of Down syndrome with Real-Time PCR method was evaluated. In this context, optimization of a suitable method for purification of high quality DNA from amniotic fluid samples was also considered. Pregnant women who had the high risk of having babies with Down syndrome were selected according to the biochemical and sonographic data and referred to the amniocentesis center. The DNA of total 59 amniotic fluid samples were extracted with different methods including boiling method, salting out method, Procedures of DNA extraction from Blood and Cell Culture by DNP_ Kit [CitmaGen], Procedure of DNA extraction from cells by DNA Isolation Kit for cells and tissues [Roche], Procedure of DNA extraction from Tissue by MagNa Pure DNA Isolation kit [Roche], and QIAamp DNA Micro Kit [Qiagen]. Then, the quality and quantity of the extracted DNA were evaluated by the NanoDrop ND- 1000 spectrophotometer device. Real-Time PCR reaction using fluorescent dye SYBR Green I [Applied Biosystems, UK] was performed to specifically amplify DSCAM and DYRK1A2 genes and the reference gene [PMP22]. Data analysis was performed using comparative cycle threshold method for the determination of the gene dosage and determining the number of copies of chromosome 21. This study showed that DNA extracted from amniotic fluid samples using QIAamp DNA Micro Kit [Qiagen] has the desirable quantity and quality for Real-Time PCR. Specific proliferation of targets and reference genes was achieved and difference between normal and affected groups based on differences between their gene dosages was determined. Prenatal diagnosis of Down syndrome is feasible by the Real-Time PCR method using DNA samples from amniotic fluid cells extracted by QIAamp DNA Micro Kit [Qiagen]. The results are comparable to the corresponding results from conventional cytogenetic methods