Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

An Exact Control‐Versus‐Treatment Comparison Test Based on Ranked Set Samples

Summary A control‐versus‐treatment multiple comparison test procedure is developed based on ranked set samples. The test, constructed based on K‐independent exact median confidence intervals corresponding to the K populations, rejects the null hypothesis of equal medians if the median confidence intervals of the control group and any one of the other K ? 1 treatment groups are disjoint. The use of the proposed test is illustrated with data from an agricultural experiment.     

Neurotoxic effect of <Emphasis Type="Italic">Caulerpa racemosa</Emphasis> var. <Emphasis Type="Italic">cylindracea</Emphasis> by neurite inhibition on the neuroblastoma cell line

In the present study, antiproliferative, apoptotic and especially neurotoxic effects of Caulerpa racemosa var. cylindracea dry and wet extracts on mouse neuroblastoma cell line, NA2B were investigated by neurotoxicity screening test (NST). C. racemosa var. cylindracea wet and dry extracts were obtained by methanol (MT) extraction. The effect of the extracts on viability and proliferation was measured by MTT. NA2B cells were induced to differentiate using 1 μM dcAMP and the amount of inhibition of growing neurites in different dilutions (50, 35, 25, 15, 10 and 5 μl/ml) by extracts was measured. The number of apoptotic cells was computed by TUNEL method using cells in culture. It was found that majority of the cells died with dry extract above the level of 15 μl/ml due to the MT effect. Below this level, on the other hand, presence of cell death and antiproliferative effect was noted due to the toxic effects of C. racemosa var. cylindracea which was independent of MT. In all doses of wet extracts, similar but less prominent dose-dependent effects were observed. Below the level of 15 μl/ml, mild toxic effect presented itself with neurite inhibition. In addition to the toxic, apoptotic and antiproliferative effects of C. racemosa var. cylindracea, its neurotoxic effects possessing property at low concentrations which manifesting itself by neurite inhibition was also showed. This species offers a potential for developing new drugs due to its antiproliferative, toxic and apoptotic effects. Nevertheless, its neurotoxic effect is a factor to be considered as multifunctional agents especially in neuronal metabolism.     

Heparanase is a predictive marker for high thrombus burden in patients with ST-segment elevation myocardial infarction

Objective: Heparanase (HPA) is an endo-β-D-glucuronidase capable of degrading heparin sulphate (HS) and heparin side chains. HPA plays a role in tumour growth, angiogenesis, cell invasion and in activation of the coagulation system. We aimed to investigate the relationship between HPA and thrombus burden (TB) in patients with ST-Segment Elevation Myocardial Infarction (STEMI).

Methods: This prospective study enrolled 187 patients with STEMI who were treated with primary percutaneous coronary intervention (pPCI). Blood samples were taken to determine serum HPA levels prior to coronary angiography and heparin administration. Serum HPA analysis was performed with a commercially available Human Elisa kit.

Results: Patients were divided into two groups: high TB (n:58) and low TB (n:129) group. Serum HPA levels were significantly higher in patients with high TB than low TB [250.1 (188.5–338.1) vs. 173.6 (134.3–219.8) pg/mL] (p?<?0.001). Serum HPA levels were higher in patients with no-reflow phenomenon compared with others [(409.3 (375.6–512.5) pg/mL vs. 186.2 (144.2–247.4) pg/mL, p?<?0.001]. In multiple logistic regression analysis HPA was a predictor of high TB.

Conclusion: Elevated HPA level in patients with STEMI is related to high TB. Furthermore, increased HPA level may be associated with thrombotic complications such as no-reflow phenomenon in patients with STEMI.     

Effect of Inoculation with Non-indigenous and Indigenous Rhizobacteria of Erzurum (Turkey) Origin on Growth and Yield of Spring Barley

Two experiments were performed to examine the effects of inoculation of barley cv. Tokak 157/37 with indigenous and non-indigenous bacterial strains. A greenhouse experiment was carried out with 75 strains isolated from Erzurum Plain and Pasinler Plain soils of Turkey, and the 6 non-indigenous strains. The 41 strains had significant positive effects on tiller number per plant, 8 strains on plant height, one strain on dry matter yield, and 24 strains on plant protein concentration. In the second experiment, the response of barley to 20 treatments (9 indigenous strains, 6 non-indigenous strains, 4 levels of N, and a control) was investigated in Erzurum Agricultural Experiment field in 2000 and 2001. Inoculation with certain indigenous and non-indigenous strains clearly benefited growth and increased the yield of field grown barley. On average of years, inoculation with Strain No. 19, Strain No. 39, Strain No. 73, Strain No. 82, BA-7, BA-142 and M-13 increased total biomass by 29.4–15.1%, grain yield by 26.6–17.7%, and total N-yield by 32.7–20.6%, as compared to the control. Indigenous Strain No. 19 was superior to all other treatments in terms of grain yield and N-yields. The higher efficacy of combining Strain No. 19 and Tokak 157/37 indicates the possibility of improved associations using olden and common cultivar and indigenous bacteria. In conclusion, Strain No. 19 seems to be suitable inoculant for barley cultivation in areas such as in Erzurum conditions.     

Focusing on the ethnobotanical uses of plants in Mersin and Adana provinces (Turkey)

This paper presents the result of a study on the herbal drugs in the herbal markets in Mersin and Adana. The data were collected through direct interviews with herbalists and customers between 2002–2005 and the popular medicinal plants were investigated. A total of 107 species belonging to 56 families were investigated and the samples were listedwith their local and Latin names. The investigation includes cross-checking the disorders and their herbal cures and their recommended use stated by the local herbalists, by the parts used, and by the preparations. The cultivated species and their ethno botanical uses, are documented and extensive inventory is presented. As a result, we observed that these plants are used especially for intestinal digestive disorders of the gastrointestinal tract, (21.68%), respiratory tract system disorders (10.43%), heart-blood circulatory system disorders (8.48%), urinary tract system disorders (7.70%), skin disorders (6.48%) and others.     

Evolution of antibiotic resistance genes: the DNA sequence of a kanamycin resistance gene from Staphylococcus aureus

The kanamycin resistance gene from Staphylococcus aureus has been sequenced and its structure compared with similar genes isolated from Streptomyces fradiae and from two transposons, Tn5 and Tn903, originally isolated from Klebsiella pneumoniae and Salmonella typhimurium, respectively. The genes are all homologous but, since their common ancestor, have undergone extensive divergence, with more than 43% divergence between the closest pair. The phylogeny of the genes cannot be made congruent to the phylogeny of the taxa from which they were isolated without requiring rather improbable differences in rates. One is therefore led to conclude that there have been multiple occurrences of gene transfer between these species. Thus, although they are homologous, they are neither orthologous nor paralogous. It is suggested that homologous genes of this type be called xenologous.