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-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α 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-α 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. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)     

Toxic effects of limonene againstCulex quinquefasciatus Say larvae and its interference with oviposition

Limonene, a major constituent of citrus essential oil isolated from abraded fresh peels of bitter orangeCitrus aurantium L., was tested for its toxicity against early 4th instar larvae ofCulex quinquefasciatus Say. The response of treated larvae was concentration-dependent and LC₉₀s were 53.80 and 32.52 ppm after 24 and 48 h of treatment respectively. Limonene treated water was less favorable for oviposition by females ofCx. quinquefasciatus.     

pH Dependent Growth of Chlorella in a Continuous Culture System 1

Production and growth of a local strain of marine Chlorella sp. was investigated under different pH levels ranging from pH 6.0–8.5 using 200 L capacity algal tubes. The results show that a pH of 6.5 will be most conducive for producing this species in a continuous culture system. The highest production was 136 g/m³/d with a mean of 117 ± 11 g/m³/d at pH 6.5 compared with other pH levels tested. The total fatty acid content was elevated at low pH levels and ω 3 PUFA increased as the pH was lowered from 8.5 to 6.5. The investigations show that algal productivity is considerably higher in the continuous culture system than in any of the other culture systems available.     

Shallow groundwater use by Safflower (Carthamus tinctorius L.) in a semi-arid region

Two-year lysimeter experiments were conducted to determine groundwater contributions by safflower (Carthamus tinctorius L.) crop. The plants were grown in twenty columns each with a diameter of 0.40 m packed with Silty Clay soil. The experiments were carried out in a complete randomized blocks design with four replicates. In each experiment, five treatments were applied by maintaining groundwater salinity to a control treatment with EC 1 dS/m, while the groundwater salinity of the other treatments was 2, 5, 8 and 10 dS/m, and 0.8 m water table level, respectively. The use of groundwater as a part of crop evapotranspiration was characterized by using daily measurements of the water level in Mariotte tubes. The extra magnitude of irrigation water requirement for each treatment was applied by water with EC of 1 dS/m. The results of experiments showed that for different control treatments with 1 dS/m, 2, 5, 8 and 10 dS/m, the groundwater contributions were achieved as 59, 51, 38, 32 and 19% of the total plant water requirements, respectively.     

Molecular survey of <Emphasis Type="Italic">Toxoplasma</Emphasis> infection in sheep and goat from Fars province,Southern Iran

Toxoplasmosis is a widespread zoonotic disease that causes significant morbidity and mortality in human fetus and in immunocompromised patients. Moreover, it becomes a major cause of abortion in sheep and goats. Since consumption of meat of infected lamb and goat is considered as the main sources of human infection in Iran, this study was undertaken to determine the prevalence of Toxoplasma infection in edible tissues of sheep and goats in Shiraz in 2008. Samples of brain, tongue, liver, and muscles of neck, intercostals, and femoral were taken from 56 sheep and 22 goats and tested by PCR. The total prevalence of Toxoplasma infection among animals was found to be 33.3%. Five out of 22 goats (22.7%) and 21 out of 56 sheep (37.5%) were infected by Toxoplasma. Differences between the prevalence rate of infection among females (nine out of 14 = 46%) and males animals (12 out of 45 = 29.5%) was significant (P = 0.013). Furthermore, a positive correlation was found between the age of animals and the rate of infection; animals greater than 2 years old showed a higher rate of infection (47%) in comparison with those less than 2 years old (25%, P = 0.04). The highest infected tissue was tongue (21.8%) followed by brain (19.2%) and femoral and intercostal muscles (17.9%). This study demonstrated a high level of Toxoplasma infection in slaughtered animals in Shiraz and these should be considered as the main sources of infection for human population in the region.     

Effects of plant residues and calcite amendments on soil sodicity

The effects of wheat, potato, sunflower, and rape residues and calcite were evaluated in soil that received sodic water. These materials were added to a sandy‐loam soil at a rate of 5%, after which the treated soils were incubated for 1 month at field‐capacity moisture and a temperature of 25°C–30°C. Column leaching experiments using treated soils were then conducted under saturated conditions using water with three sodium‐adsorption ratios (SAR) (0, 10, 40) with a constant ionic strength (50 mmol L^–1). The results indicated that the application of plant residues to soils caused an increase in cation‐exchange capacity and exchangeable cations. Leaching experiments indicated that the addition of plant amendments led to increased Na⁺ leaching and decrease in exchangeable‐sodium percentage (ESP). The ESP of the control soil, after leaching with solutions with an SAR of 10 and 40, increased significantly, but the level of sodification in soils treated with plant residue was lower. Such decreases of soil ESP were greatly affected by the type of plant residues, with the order of: potato‐treated soil > sunflower‐treated soil > rape‐treated soil > wheat‐treated soil > calcite‐treated soil > control soil.     

A nonpathogenic species of binucleate Rhizoctonia inhibits the formation of infection structures caused by Rhizoctonia solani on cucumber

Binucleate Rhizoctonia (BNR) W7 was evaluated for the efficacy to control damping-off disease in cucumber seedlings. Results revealed that Lobate appresoria and dome-shaped infection cushion were produced by virulent Rhizoctonia solani C9 AG4 on the surface of the cucumber seedling hypocotyls, causing damping-off, 12 h after inoculation. A significant positive correlation was observed between disease severity rating and number of infection cushions (r = 0.94) and appresoria (r = 0.97) on the hypocotyls of seedlings inoculated with virulent R. solani C9 alone. Inoculation of a nonpathogenic species of BNR prior to virulent R. solani significantly reduced the number of appresoria on hypocotyls surface and inhibited the formation of infection cushions that accompanied by highly significant (P ≤ 0.01) reduction in disease severity in comparison with seedling inoculated with R. solani C9 alone. Formation of mucilaginous material upon recognition of the nonpathogenic isolate of BNR followed by hyphal lysis of BNR W7 6 h after inoculation was responsible for reduction in appresorium formation and complete inhibition of infection cushion formation. However, the number of infection structures produced on cucumber was not significantly different for hypocotyls of seedlings inoculated with R. solani C9 with and without the nonpathogenic BNR W7.