Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Human immune response against salivary antigens of Simulium damnosum s.l.: A new epidemiological marker for exposure to blackfly bites in onchocerciasis endemic areas

BackgroundSimulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated.Methodology/Principal findingsBlood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC–MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC–MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase.Conclusions/SignificanceThis study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.     

Risk of venous thromboembolism in women with polycystic ovary syndrome: a population-based matched cohort analysis

Background:

There is an increased risk of venous thromboembolism among women taking oral contraceptives. However, whether there is an additional risk among women with polycystic ovary syndrome (PCOS) is unknown.

Methods:

We developed a population-based cohort from the IMS LifeLink Health Plan Claims Database, which includes managed care organizations in the United States. Women aged 18–46 years taking combined oral contraceptives and who had a claim for PCOS (n = 43 506) were matched, based on a propensity score, to control women (n = 43 506) taking oral contraceptives. Venous thromboembolism was defined using administrative coding and use of anticoagulation. We used Cox proportional hazards models to assess the relative risk (RR) of venous thromboembolism among users of combined oral contraceptives with and without PCOS.

Results:

The incidence of venous thromboembolism among women with PCOS was 23.7/10 000 person-years, while that for matched controls was 10.9/10 000 person-years. Women with PCOS taking combined oral contraceptives had an RR for venous thromboembolism of 2.14 (95% confidence interval [CI] 1.41–3.24) compared with other contraceptive users. The incidence of venous thromboembolism was 6.3/10 000 person-years among women with PCOS not taking oral contraceptives; the incidence was 4.1/10 000 person-years among matched controls. The RR of venous thromboembolism among women with PCOS not taking oral contraceptives was 1.55 (95% CI 1.10–2.19).

Interpretation:

We found a 2-fold increased risk of venous thromboembolism among women with PCOS who were taking combined oral contraceptives and a 1.5-fold increased risk among women with PCOS not taking oral contraceptives. Physicians should consider the increased risk of venous thromboembolism when prescribing contraceptive therapy to women with PCOS.Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age. The National Institutes of Health criteria estimates its prevalence in the United States to be between 6% and 10%, while the Rotterdam criteria estimates the prevalence to be as high as 15%.¹ Although its cause is not entirely known, the diagnostic criteria include oligo- or anovulation, clinical and/or biochemical signs of hyperandrogenism, and polycystic ovaries.² Women often present with clinical manifestations of high androgen levels, including facial hair growth (hirsutism), acne vulgaris and hair loss on the scalp. Previous studies reported the prevalence of impaired glucose tolerance to be 31.1%–35.2% and the prevalence of type 2 diabetes to be 7.5%–9.8% among women with PCOS.^3,4 A recent consensus workshop reported that the prevalence of several known risk factors for cardiovascular disease (hypertension, diabetes, abdominal obesity, psychological factors, smoking, altered apoA1/ApoB ratios) are doubled among women with PCOS compared with matched controls.^1,5Combined oral contraceptives are the mainstay treatment for PCOS. However, they are also known to elevate the risk of venous thromboembolism and cardiovascular disease.⁶ To date, contraceptive studies involving women with PCOS have focused mainly on efficacy, evaluating the effect of combined oral contraceptives on the reduction of hirsutism and hyperandrogenism.^7,8 Two studies assessed the metabolic effects of combined oral contraceptives in PCOS, but these studies had small sample sizes and could not evaluate for cardiovascular events.^9,10Although women with PCOS have an increase in both cardiovascular risk factors and subclinical cardiovascular disease,¹¹ recent guidelines have concluded there are no data in the literature assessing the association between the use of oral contraceptives and cardiovascular disease among women with PCOS.² Because combined oral contraceptives are the mainstay treatment, our objective was to determine whether women with PCOS taking combined oral contraceptives have a greater risk of venous thromboembolism compared with other contraceptive users. We also examined whether women with PCOS not taking oral contraceptives had an increased risk of venous thromboembolism compared with the general population.     

58 Packaging trinucleotide repeats: the effect of CAG/CTG repeats on nucleosome formation

In neurological diseases such as fragile X syndrome, spinal and bulbar muscular atrophy, myotonic dystrophy, and Huntington’s disease, the molecular basis of pathogenicity is the presence of an expanded trinucleotide repeat (TNR) tract (Ashley & Warren, 1995). TNRs implicated in many of these diseases are composed of CAG/CTG repeats. For example, in healthy individuals 5–35, CAG/CTG TNR repeats are present in the huntingtin gene. However, individuals with 40 or greater repeats will develop Huntington’s disease (Andrew et al., 1993). We are particularly interested in how these TNR sequences are packaged in chromatin. Recent evaluations of CAG/CTG TNR sequences in our laboratory have demonstrated that the repeats increase the propensity for the DNA sequences to incorporate into nucleosomes, where nucleosomes represent the minimal unit of packaging in chromatin (Volle & Delaney, 2012). In this work, we are interested in determining the minimum number of CAG/CTG repeats required to confer a significant increase in nucleosome incorporation relative to sequences that lack the TNR sequence. By defining the changes imposed on these fundamental interactions by the presence of a CAG/CTG repeat tract, we will gain insight into the possible interactions that allow for the expansion of these TNR tracts.     

Effects of reduced seawater pH on fertilisation, embryogenesis and larval development in the Antarctic seastar Odontaster validus

The effects of ocean acidification will be pronounced in high-latitude marine communities, although little is known on how reproduction in free-spawning polar invertebrates will respond. Using the circum-Antarctic sea star Odontaster validus, we examined fertilisation, larval survival and development under a controlled seawater treatment (temperature = ?0.5 °C, pH 8.1, pCO₂(aq) = 326.6 μatm, TA = 2,274.2 μmol kg soln^?1), two near-future pH treatments (pH 7.8 and 7.6) and an extreme treatment (pH 7.0). At a sperm concentration of 3.5 × 10⁵ sperm ml^?1, percentage of fertilisation was 98–90 % across a pH 8.1–7.0 range. At near-future pH ranges (pH 7.8 and 7.6), fertilisation was not significantly lower than in the control pH 8.1 except at the lowest sperm concentration (2.2 × 10³ sperm ml^?1) where fertilisation was reduced to 60 and 61 % in pH 7.6 and 7.8, respectively. Larval survival was not significantly affected by a decrease in pH of 0.3 units, but at pH 7.6 survival was significantly reduced. This difference was apparent at 9 days, and at the end of the experiment at 58 days, survival was 55 % compared with 85 % in the ambient treatment. Near-future changes to pH yielded smaller larvae, a result of both subtle differences in their morphology and slowed development rates, while larvae at pH 7.0 showed evidence of abnormal development. O. validus fertilisation and larval success declines in seawater pH conditions expected in coastal Antarctica over the coming decades, although the responses observed are within the range observed in warmer-water echinoderms.     

Soil test measures of available P (Colwell, resin and DGT) compared with plant P uptake using isotope dilution

Background and aims

Recent research has demonstrated the high accuracy of a new method for assessment of plant available P in soil called diffusive gradients in thin-films (DGT). The process of P released by additions of bicarbonate to soil samples simulating common soil P tests is yet to be assessed by the new method (DGT). The aim of this study was to identify the pools of soil P extracted by soil test methods (DGT, Colwell and resin) by comparing, in ³²P–labelled soils, the specific activity (SA) of phosphorus extracted by common soil test extracts with the SA of wheat plants grown in a range of agricultural soils from southern Australia.

Methods

Wheat (cv. Frame) was grown for 4 weeks in 14 soils that were labelled uniformly with carrier-free ³²P. The specific activity (SA) of P (MBq ³²P kg ³¹P^?1) in each soil test extract was compared to the SA of P in the wheat plants.

Results

The SA of P in plants were similar to P extracted by the Colwell extractant in only 4 of the 14 soils; while SA in plants and extractants corresponded in 10 of the soils for the resin method and in 12 of the soils for the DGT method. Phosphorus in the Colwell and resin extract solutions had significantly lower SAs compared to P in the plants for 10 and 4 of the soils, respectively, indicating greater extraction of non-labile P sources (unlabelled ³¹P). Phosphorus in the DGT extractant had significantly lower SA than the plants for 1 soil and in 1 soil the SA was higher. Overall, across all soils, 25 % of P extracted by the Colwell method was non labile compared to 9 % and 2 % for the resin and DGT methods, respectively.

Conclusion

The new DGT method for extraction of soil P has the potential to accurately predict occurrences of P deficiency because it generally extracts the same pool of labile soil P accessed by wheat plants, while methods using bicarbonate solution (e.g. Colwell, Olsen) or water (resin) at wide soil:solution ratios are more likely to measure more non-labile forms of P in soil.     

Host mTORC1 Signaling Regulates Andes Virus Replication

Hantavirus pulmonary syndrome (HPS) is a severe respiratory disease characterized by pulmonary edema, with fatality rates of 35 to 45%. Disease occurs following infection with pathogenic New World hantaviruses, such as Andes virus (ANDV), which targets lung microvascular endothelial cells. During replication, the virus scavenges 5′-m⁷G caps from cellular mRNA to ensure efficient translation of viral proteins by the host cell cap-dependent translation machinery. In cells, the mammalian target of rapamycin (mTOR) regulates the activity of host cap-dependent translation by integrating amino acid, energy, and oxygen availability signals. Since there is no approved pharmacological treatment for HPS, we investigated whether inhibitors of the mTOR pathway could reduce hantavirus infection. Here, we demonstrate that treatment with the FDA-approved rapamycin analogue temsirolimus (CCI-779) blocks ANDV protein expression and virion release but not entry into primary human microvascular endothelial cells. This effect was specific to viral proteins, as temsirolimus treatment did not block host protein synthesis. We confirmed that temsirolimus targeted host mTOR complex 1 (mTORC1) and not a viral protein, as knockdown of mTORC1 and mTORC1 activators but not mTOR complex 2 components reduced ANDV replication. Additionally, primary fibroblasts from a patient with tuberous sclerosis exhibited increased mTORC1 activity and increased ANDV protein expression, which were blocked following temsirolimus treatment. Finally, we show that ANDV glycoprotein Gn colocalized with mTOR and lysosomes in infected cells. Together, these data demonstrate that mTORC1 signaling regulates ANDV replication and suggest that the hantavirus Gn protein may modulate mTOR and lysosomal signaling during infection, thus bypassing the cellular regulation of translation.     

Effects of wildfire, rainfall and region on desert lizard assemblages: the importance of multi-scale processes

Vertebrate populations are influenced by environmental processes that operate at a range of spatial and temporal scales. Wildfire is a disturbance that can affect vertebrate populations across large spatial scales, although vertebrate responses are frequently influenced by processes operating at smaller spatial scales such as topography, interspecific interactions and regional history. Here, we investigate the effects of a broad-scale wildfire on lizard assemblages in a desert region. We predicted that a rainfall gradient within the region affected by the wildfire would influence lizard responses to the fire by encouraging post-fire succession to proceed more rapidly in high-rainfall areas, and would be enabled in turn by more rapid vegetation recovery. To test our prediction, we censused lizards, measured rainfall, undertook vegetation surveys and sampled invertebrate abundance across burnt and unburnt habitat ecotones within three regional areas situated along a gradient of long-term annual rainfall. Lizard diversity was not affected by fire or region and lizard abundance was influenced only by region. Lizard assemblage composition was also only influenced by region, but this did not relate to differences in rainfall or habitat as we had predicted. Regional differences in lizard assemblages related instead to food availability. The observed differences also likely reflected regional differences in the strength of biotic interactions with predators and changes in land use. Our study shows that assemblage responses to a disturbance were not uniform within a large desert region and instead were influenced by other environmental processes operating simultaneously at multiple temporal and spatial scales.     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.