C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex

Background  

Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex.     

Biodistribution of gyroxin using 125I as radiotracer

Summary The use of radiotracers in the research of animal venom has been scarce, although it allows an excellent approach to follow the process of bioavailability, biodistribution and kinetics of toxins. The purpose of this study was to assess gyroxin action mechanism, transport, compartments and action sites. This toxin is a thrombin-like and causes the barrel rotation syndrome. The gyroxin was labeled with ¹²⁵I and used as a tracer for the in vivo assay in mice. Blood samples and organs were collected at different time intervals, weighed and analyzed in a gamma-counter. The data was related with tissues distribution of protease activated receptor (PAR). Biodistribution assay allowed dividing the organs into three groups. The first one with the organs that followed the blood kinetics, the second with the organs related to metabolisms and elimination, and the third with the organs in which the gyroxin concentration increased during the observation period.     

Enhancement of Human Thyrotropin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

The influence of sodium butyrate (NaBu) on the synthesis of recombinant human thyrotropin (r-hTSH) by CHO cells was investigated for the first time. A volumetric productivity of ~10 μg hTSH/mL was repeatedly obtained, with a 3.3-fold increase over a control culture carried out in the absence of NaBu. Since NaBu can induce CHO cell apoptosis and cell growth arrest, the increase in specific productivity was even higher, i.e., ca. 5-fold. Analysis of the N-glycan composition of r-hTSH obtained with the addition of NaBu to the culture medium showed an approximately 12 % increase in the amount of sialic acid, as well as in total carbohydrate, partly due to the increase in the site occupancy from 2.77 to 2.93 glycans per mole of hTSH. The two hormone preparations were characterized by N-glycan structural analysis, which showed that NaBu increased the bi-antennary structures by ca. 13 % while decreasing the tri-antennary structures by approximately the same amount. The in vivo biological activity and pharmacokinetic behavior (clearance) were found to be similar for the two hormone preparations.     

Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (°C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 °C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 × 10⁶ cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (∼80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (∼9 × 10⁵ cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce ∼1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.     

Validation of a stability-indicating RP-LC method for the assessment of recombinant human interleukin-11 and its correlation with bioassay

A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11), based on the ICH guidelines. The method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 25°C. The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using a photodiode array (PDA) detection at 214 nm. Separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1-200 μg/mL (r(2) = 0.9995). Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p > 0.05). The method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay.     

Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 °C in acetic acid. The α-and β-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of >98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C₄ column (80–90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.     

High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space.

A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.