LC-MS characterization and purity assessment of a prototype bispecific antibody

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MS^E peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection.     

Controlling Hierarchy in Solution‐processed Polymer Solar Cells Based on Crosslinked P3HT

Understanding and controlling the morphology of donor/acceptor blends is critical for the development of solution processable organic solar cells. By crosslinking a poly(3‐n‐hexylthiophene‐2,5‐diyl) (P3HT) film we have been able to spin‐coat [6,6]‐phenyl‐C₆₁‐butyric acid methyl ester (PCBM) onto the film to form a structure that is close to a bilayer, thus creating an ideal platform for investigating interdiffusion in this model system. Neutron reflectometry (NR) demonstrates that without any thermal treatment a smaller amount of PCBM percolates throughout the crosslinked P3HT when compared to a non‐crosslinked P3HT film. Using time‐resolved NR we also show thermal annealing increases the rate of diffusion, resulting in a near‐uniform distribution of PCBM throughout the polymer film. XPS measurements confirm the presence of both P3HT and PCBM at the annealed film's surface indicating that the two components are intermixed. Photovoltaic devices fabricated using this bilayer approach and suitable annealing conditions yielded comparable power conversion efficiencies to bulk heterojunction devices made from the same materials. The crosslinking procedure has also enabled the formation of patterned P3HT films by photolithography. Pillars with feature sizes down to 2 μm were produced and after subsequent deposition of PCBM and thermal annealing devices with efficiencies of up to 1.4% were produced.     

Rapid Sequential Spread of Two Wolbachia Variants in Drosophila simulans

The maternally inherited intracellular bacteria Wolbachia can manipulate host reproduction in various ways that foster frequency increases within and among host populations. Manipulations involving cytoplasmic incompatibility (CI), where matings between infected males and uninfected females produce non-viable embryos, are common in arthropods and produce a reproductive advantage for infected females. CI was associated with the spread of Wolbachia variant wRi in Californian populations of Drosophila simulans, which was interpreted as a bistable wave, in which local infection frequencies tend to increase only once the infection becomes sufficiently common to offset imperfect maternal transmission and infection costs. However, maternally inherited Wolbachia are expected to evolve towards mutualism, and they are known to increase host fitness by protecting against infectious microbes or increasing fecundity. We describe the sequential spread over approximately 20 years in natural populations of D. simulans on the east coast of Australia of two Wolbachia variants (wAu and wRi), only one of which causes significant CI, with wRi displacing wAu since 2004. Wolbachia and mtDNA frequency data and analyses suggest that these dynamics, as well as the earlier spread in California, are best understood as Fisherian waves of favourable variants, in which local spread tends to occur from arbitrarily low frequencies. We discuss implications for Wolbachia-host dynamics and coevolution and for applications of Wolbachia to disease control.     

Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, > 500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4′-methyl-N²-3-pyridinyl-4,5′-bi-1,3-thiazole-2,2′-diamine (JNK Docking (JD) compound 123, but not the related compound (4′-methyl-N ~ 2 ~ -(6-methyl-2-pyridinyl)-4,5′-bi-1,3-thiazole-2,2′-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.     

Role of Baseline Antral Follicle Count and Anti-Mullerian Hormone in Prediction of Cumulative Live Birth in the First In Vitro Fertilisation Cycle: A Retrospective Cohort Analysis

Objective

This retrospective study determined for the first time the role of baseline antral follicle count (AFC) and serum anti-Mullerian hormone (AMH) level in the first in-vitro fertilisation (IVF) cycle in predicting cumulative live birth from one stimulation cycle.

Methods

We studied 1,156 women (median age 35 years) undergoing the first IVF cycle. Baseline AFC and AMH level on the day before ovarian stimulation were analysed. The main outcome measure was cumulative live birth in the fresh plus all the frozen embryo transfers after the same stimulation cycle.

Results

Serum AMH was significantly correlated with AFC. Both AMH and AFC showed significant correlation with age and ovarian response in the stimulated cycle and total number of transferrable embryos. Baseline AFC and serum AMH were significantly higher in subjects attaining a live birth than those who did not in the fresh stimulated cycle, as well as those attaining cumulative live birth. There was a significant trend of higher cumulative live birth rate in women with higher AMH or AFC. However, logistic regression revealed that both AMH and AFC were not significant predictors of cumulative live birth after adjusting for age and number of embryos available for transfer. Considering only one single predictor, the areas under the ROC curves for AMH (0.646, 95% CI 0.616–0.675) and age (0.648, 95% CI 0.618–0.677) were slightly higher than that for AFC (0.617, 95% CI 0.587–0.647) in predicting cumulative live birth. However, a model combining AMH (with or without AFC) and age of the women only classified an addition of less than 2% of subjects correctly compared to the model with age alone.

Conclusion

Baseline AFC and serum AMH have only modest predictive performance on the occurrence of cumulative live birth, and may not give additional value on top of the women''s age.     

Endometrial Exosomes/Microvesicles in the Uterine Microenvironment: A New Paradigm for Embryo-Endometrial Cross Talk at Implantation

Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100–300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50–150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.     

Measuring Airway Surface Liquid Depth in Ex Vivo Mouse Airways by X-Ray Imaging for the Assessment of Cystic Fibrosis Airway Therapies

In the airways of those with cystic fibrosis (CF), the leading pathophysiological hypothesis is that an ion channel defect results in a relative decrease in airway surface liquid (ASL) volume, producing thick and sticky mucus that facilitates the establishment and progression of early fatal lung disease. This hypothesis predicts that any successful CF airway treatment for this fundamental channel defect should increase the ASL volume, but up until now there has been no method of measuring this volume that would be compatible with in vivo monitoring. In order to accurately monitor the volume of the ASL, we have developed a new x-ray phase contrast imaging method that utilizes a highly attenuating reference grid. In this study we used this imaging method to examine the effect of a current clinical CF treatment, aerosolized hypertonic saline, on ASL depth in ex vivo normal mouse tracheas, as the first step towards non-invasive in vivo ASL imaging. The ex vivo tracheas were treated with hypertonic saline, isotonic saline or no treatment using a nebuliser integrated within a small animal ventilator circuit. Those tracheas exposed to hypertonic saline showed a transient increase in the ASL depth, which continued for nine minutes post-treatment, before returning to baseline by twelve minutes. These findings are consistent with existing measurements on epithelial cell cultures, and therefore suggest promise for the future development of in vivo testing of treatments. Our grid-based imaging technique measures the ASL depth with micron resolution, and can directly observe the effect of treatments expected to increase ASL depth, prior to any changes in overall lung health. The ability to non-invasively observe micron changes in the airway surface, particularly if achieved in an in vivo setting, may have potential in pre-clinical research designed to bring new treatments for CF and other airway diseases to clinical trials.     

Trends in the Incidence of In Situ and Invasive Cervical Cancer by Age Group and Histological Type in Korea from 1993 to 2009

Objective

Our study aims to describe changes in carcinoma in situ (CIS) and invasive cervical carcinoma (ICC) in Korean women diagnosed between 1993 and 2009.

Methods

All cases of CIS and invasive cervical carcinoma diagnosed from 1993 to 2009 in the Korean National Cancer Incidence database were analyzed. Age-standardized rates (ASRs) and annual percent changes (APCs) in incidence rates were compared according to age and histological type. Additionally, we used Korea National Health and Nutrition Examination Survey (KNHANES) to know the association between screening rate for cervical cancer and incidence rate of cervical cancer.

Results

Between 1993 and 2009, 72,240 cases of ICC were reported in Korea. Total incidence rate of ICC was 14.7 per 100,000 females. ASRs of ICC declined 3.8% per year, from 19.3 per 100,000 in 1993 to 10.5 per 100,000 in 2009. Although the overall incidence rate of adenocarcinoma remained stable, invasive squamous cell carcinoma showed a decreasing trend (APC −4.2%). For women aged 60–79 years, ASRs for squamous cell carcinoma increased from 1993 to 2001, and decreased from 2001 to 2009 (APC: −4.6%). Total 62,300 cases of CIS were diagnosed from 1993 to 2009. Total incidence rate of CIS was 12.2 per 100,000 females. ASRs of CIS increased 5.7% per year, from 7.5 per 100,000 in 1993 to 19.0 per 100,000 in 2009. Adenocarcinoma in situ increased 13.2% per year. There was a strong positive correlation between screening rate for cervical cancer and incidence rate for CIS (p-value = 0.03) whereas screening rate showed a strong negative correlation with incidence rate for squamous ICC (p-value = 0.04).

Conclusions

The increasing trend in CIS, coupled with a decreasing trend in ICC, suggests the important role of cervix cancer screening. The incidence of adenocarcinoma showed a plateau, but the incidence of adenocarcinoma in situ showed an increasing trend.