Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate <i>in Vivo</i> Deamidation of Therapeutic Antibodies

Deamidation of therapeutic antibodies may result in decreased drug activity and undesirable changes in pharmacokinetics and immunogenicity. Therefore, it is necessary to monitor the deamidation levels [during storage] and after <i>in vivo</i> administration. Because of the complexity of <i>in vivo</i> samples, immuno-affinity capture is widely used for specific enrichment of the target antibody prior to LC–MS. However, the conventional use of bead-based methods requires large sample volumes and extensive processing steps. Furthermore, with automation difficulties and extended sample preparation time, bead-based approaches may increase artificial deamidation. To overcome these challenges, we developed an automated platform to perform tip-based affinity capture of antibodies from complex matrixes with rapid digestion and peptide elution into 96-well microtiter plates followed by LC–MS analysis. Detailed analyses showed that the new method presents high repeatability and reproducibility with both intra and inter assay CVs < 8%. Using the automated platform, we successfully quantified the levels of deamidation of a humanized monoclonal antibody in cynomolgus monkeys over a time period of 12 weeks after administration. Moreover, we found that deamidation kinetics between <i>in vivo</i> samples and samples stressed <i>in vitro</i> at neutral pH were consistent, suggesting that the <i>in vitro</i> stress test may be used as a method to predict the liability to deamidation of therapeutic antibodies <i>in vivo</i>

Fe and Zn-loaded cotton-sticks biochar and organic amendments improves chickpea production by enhancing ionic uptake and chemical properties of sandy soils

Low nutrient availability in sandy soils is a serious threat to crop productivity. The present study evaluated the effectiveness of cotton-sticks biochar, sugarcane press-mud, and vegetable compost on soil chemical properties and chickpea yield in sandy soils. Two chickpea varieties, Bittal-2016 and Noor-2019, were grown in soil amended with cotton-sticks biochar at 10 and 15 Mg ha−1; sugarcane press-mud at 10 and 15 Mg ha−1; vegetable compost at 10 and 15 Mg ha−1; cotton-sticks biochar at 10 and 15 Mg ha−1 plus loaded with Fe/Zn including no amendment. Cotton-sticks biochar at 15 Mg ha−1 loaded with Fe/Zn significantly increased chlorophyll (48.40), N (3.1, 2.5, and 3.9%), P (1.13%, 1.10, and 1.4%), and K (1.44, 2.26, and 1.27%) in root, shoot, and grains of var. Noor-2019. Soil amended with vegetable compost at 10 Mg ha−1 and Fe/Zn-loaded biochar at 15 Mg ha−1 exhibited a marked increase in shoot length (43.2 cm), root length (25.8 cm), root fresh weight (8.0 g), pods per plant (34), 100-grain weight (32.7 g), and grain yield (25.9 g) for var. Noor-2019. Amended soil with Fe/Zn-loaded biochar at 10 Mg ha−1 depicted a remarkable increase in soil EC, pH, OM, and N by 2.12 dS m−1, 8.4, 0.67% and 0.20%, respectively. Conclusively, Fe/Zn-loaded cotton-sticks biochar at 15 Mg ha−1 and vegetable compost at 10 Mg ha−1 effectively improved the soil chemical properties, nutrient uptake, and yield of var. Noor-2019. Hence, cotton-sticks biochar could be used as eco-friendly approach for improving chickpea production in coarse-textured soils.</p

Four Genetic Polymorphisms of Lymphotoxin-Alpha Gene and Cancer Risk: A Systematic Review and Meta-Analysis

<div><p>Lymphotoxin-alpha (LTA) is a pro-inflammatory cytokine that plays an important role in the inflammatory and immunologic response. Numerous studies have shown <i>LTA</i> polymorphisms as risk factors for cancers, but the results remain inconclusive. The goal of the present meta-analyses is to establish the associations between cancers and four <i>LTA</i> variants (rs1041981, rs2239704, rs2229094 and rs746868). A total of 30 case-control studies involving 58,649 participants were included in the current meta-analyses. Our results showed significant associations with increased cancer risk for rs1041981 (odd ratio (OR) = 1.15, 99% confidential interval (CI) = 1.07-1.25, P < 0.0001, I² = 12.2%), rs2239704 (OR = 1.08, 99% CI = 1.01-1.16, P = 0.021, I² = 0.0%) and rs2229094 (OR = 1.28, 99% CI = 1.09-1.50, P = 0.003, I² = 0.0%). No evidence was found for the association between rs746868 and cancer risk (OR = 1.01, 99% CI = 0.93-1.10, P = 0.771, I² = 0.0%). Subgroup meta-analysis suggested that rs2239704 was likely to increase the risk of hematological malignancy (OR = 1.10, 99% CI = 1.01–1.20, P = 0.023, I² = 0.0%), and rs2229094 was specific for the increased risk of adenocarcinoma (OR = 1.33, 99% CI = 1.11-1.59, P = 0.002, I² = 0.0%). In conclusion, our meta-analyses suggested that the <i>LTA</i> rs1041981, rs2239704 and rs2229094 polymorphisms contributed to the increased risk of cancers. Future functional studies were needed to clarify the mechanistic roles of the three variants in the cancer risk.</p> </div

Meta-analysis of <i>LTA</i> rs2229094 polymorphism and cancer risk stratified by cancer type.

<p>Meta-analysis of <i>LTA</i> rs2229094 polymorphism and cancer risk stratified by cancer type.</p

Meta-analysis of <i>LTA</i> rs1041981 polymorphism and cancer risk in the additive model stratified by cancer type.

<p>Meta-analysis of <i>LTA</i> rs1041981 polymorphism and cancer risk in the additive model stratified by cancer type.</p

Flow diagram depicts literature search and study selection.

<p>Flow diagram depicts literature search and study selection.</p

Funnel plots of <i>LTA</i> polymorphisms and cancer risk data for publication bias.

<p>Funnel plots of <i>LTA</i> polymorphisms and cancer risk data for publication bias.</p