Learning-Based Non-Intrusive Electric Load Monitoring for Smart Energy Management.

State-of-the-art smart cities have been calling for economic but efficient energy management over a large-scale network, especially for the electric power system. It is a critical issue to monitor, analyze, and control electric loads of all users in the system. In this study, a non-intrusive load monitoring method was designed for smart power management using computer vision techniques popular in artificial intelligence. First of all, one-dimensional current signals are mapped onto two-dimensional color feature images using signal transforms (including the wavelet transform and discrete Fourier transform) and Gramian Angular Field (GAF) methods. Second, a deep neural network with multi-scale feature extraction and attention mechanism is proposed to recognize all electrical loads from the color feature images. Third, a cloud-based approach was designed for the non-intrusive monitoring of all users, thereby saving energy costs during power system control. Experimental results on both public and private datasets demonstrate that the method achieves superior performances compared to its peers, and thus supports efficient energy management over a large-scale Internet of Things network.

A genome-wide CRISPR screen identifies USP1 as a novel regulator of the mammalian circadian clock.

The circadian clock is generated by a molecular timekeeping mechanism coordinating daily oscillations of physiology and behaviors in mammals. In the mammalian circadian clockwork, basic helix-loop-helix ARNT-like protein 1 (BMAL1) is a core circadian component whose defects lead to circadian disruption and elicit behavioral arrhythmicity. To identify previously unknown regulators for circadian clocks, we searched for genes influencing BMAL1 protein level by using a CRISPR/Cas9-based genome-wide knockout library. As a result, we found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1 (USP1) positively affects BMAL1 protein abundance. Overexpression of wild-type USP1, but not a deubiquitinase-inactive mutant USP1, upregulated BMAL1 protein level, whereas genetic ablation of USP1 downregulated BMAL1 protein level in U2OS cells. Furthermore, treatment with USP1 inhibitors led to significant downregulation of BMAL1 protein in U2OS cells as well as mouse tissues. Subsequently, genetic ablation or pharmacological inhibition of USP1 resulted in reduced mRNA levels of a panel of clock genes and disrupted circadian rhythms in U2OS cells. Mechanistically, USP1 was able to de-ubiquitinate BMAL1 and inhibit the proteasomal degradation of BMAL1. Interestingly, the expression of Usp1 was much higher than the other two deubiquitinases of BMAL1 (Usp2 and Usp9X) in the mouse heart, implying a tissue-specific function of USP1 in the regulation of BMAL1 stability. Our work thus identifies deubiquitinase USP1 as a previously unknown regulator of the mammalian circadian clock and highlights the potential of genome-wide CRISPR screens in the identification of regulators for the circadian clock.

Atypical Asparagine Deamidation of NW Motif Significantly Attenuates the Biological Activities of an Antibody Drug Conjugate.

Asparagine deamidation is a post-translational modification (PTM) that converts asparagine residues into iso-aspartate and/or aspartate. Non-enzymatic asparagine deamidation is observed frequently during the manufacturing, processing, and/or storage of biotherapeutic proteins. Depending on the site of deamidation, this PTM can significantly impact the therapeutic's potency, stability, and/or immunogenicity. Thus, deamidation is routinely monitored as a potential critical quality attribute. The initial evaluation of an asparagine's potential to deamidate begins with identifying sequence liabilities, in which the n + 1 amino acid is of particular interest. NW is one motif that occurs frequently within the complementarity-determining region (CDR) of therapeutic antibodies, but according to the published literature, has a very low risk of deamidating. Here we report an unusual case of this NW motif readily deamidating within the CDR of an antibody drug conjugate (ADC), which greatly impacts the ADC's biological activities. Furthermore, this NW motif solely deamidates into iso-aspartate, rather than the typical mixture of iso-aspartate and aspartate. Interestingly, biological activities are more severely impacted by the conversion of asparagine into iso-aspartate via deamidation than by conversion into aspartate via mutagenesis. Here, we detail the discovery of this unusual NW deamidation occurrence, characterize its impact on biological activities, and utilize structural data and modeling to explain why conversion to iso-aspartate is favored and impacts biological activities more severely.

Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

CMC Regulatory Considerations for Antibody-Drug Conjugates.

Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included.

Cry1Δ11 mutation induces ADHD-like symptoms through hyperactive dopamine D1 receptor signaling.

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder that affects approximately 5.3% of children and approximately 2.5% of adults. There is an intimate relationship between ADHD and sleep disturbance. Specifically, individuals carry a mutation in the core circadian gene CRY1 (c. 1657 + 3A > C), which results in the deletion of exon 11 expression in the CRY1 protein (CRY1Δ11), causing them to exhibit typical ADHD symptoms. However, the underlying mechanism is still elusive. In this study, we demonstrate that Cry1Δ11 (c. 1717 + 3A > C) mice showed ADHD-like symptoms, including hyperactivity, impulsivity, and deficits in learning and memory. A hyperactive cAMP signaling pathway was found in the nucleus accumbens (NAc) of Cry1Δ11 mice. We further demonstrated that upregulated c-Fos was mainly localized in dopamine D1 receptor-expressing medium spiny neurons (DRD1-MSNs) in the NAc. Neuronal excitability of DRD1-MSNs in the NAc of Cry1Δ11 mice was significantly higher than that of WT controls. Mechanistically, the CRY1Δ11 protein, in contrast to the WT CRY1 protein, failed to interact with the Gαs protein and inhibit DRD1 signaling. Finally, the DRD1 antagonist SCH23390 normalized most ADHD-like symptoms in Cry1Δ11 mice. Thus, our results reveal hyperactive DRD1 signaling as an underlying mechanism and therapeutic target for ADHD induced by the highly prevalent CRY1Δ11 mutation.

FMRP binds Per1 mRNA and downregulates its protein expression in mice.

FMRP, an RNA-binding protein, has previously shown to be involved in regulation of circadian rhythms in flies and mice. However, the molecular mechanism remains elusive. Here we demonstrate that core circadian component Per1 mRNA was a target of FMRP and the association leads to reduced PER1 expression. In Fmr1 KO mice, the oscillation of PER1 protein expression was significantly affected in a temporal and tissue-dependent pattern when compared to WT mice. Our work thus identified Per1 mRNA as a novel target of FMRP and suggested a potential role of FMRP in regulation of circadian function.

Characterization of structures and molecular interactions of RNA and lipid carriers using atomic force microscopy.

Ribonucleic acid (RNA) and lipid are essential biomolecules in many biological processes, and hold a great prospect for biomedical applications, such as gene therapy, vaccines and therapeutic drug delivery. The characterization of morphology and intra-/inter-molecular interactions of RNA and lipid molecules is critical for understanding their functioning mechanisms. Atomic force microscopy (AFM) is a sophisticated technique for characterizing biomolecules featured by its piconewton force sensitivity, sub-nanometer spatial resolution, and flexible operation conditions in both air and liquid. The goal of this review is to highlight the representative and outstanding discoveries of the characterization of RNA and lipid molecules through morphology identification, physicochemical property determination and intermolecular force measurements by AFM. The first section introduces the AFM imaging of RNA molecules to obtain high-resolution morphologies and nanostructures in air and liquid, followed by the discussion of employing AFM force spectroscopy in understanding the nanomechanical properties and intra-/inter-molecular interactions of RNA molecules, including RNA-RNA and RNA-biomolecule interactions. The second section focuses on the studies of lipid and RNA encapsulated in lipid carrier (RNA-lipid) by AFM as well as the sample preparation and factors influencing the morphology and structure of lipid/RNA-lipid complexes. Particularly, the nanomechanical properties of lipid and RNA-lipid characterized by nanomechanical imaging and force measurements are discussed. The future perspectives and remaining challenges on the characterization of RNA and lipid offered by the versatile AFM techniques are also discussed. This review provides useful insights on the characterization of RNA and lipids nanostructures along with their molecular interactions, and also enlightens the application of AFM techniques in investigating a broad variety of biomolecules.

A randomized controlled trial: evaluation of efficiency and safety of a novel surgical guide in the extraction of deeply impacted supernumerary teeth in the anterior maxilla.

Background: Preoperative X-ray and cone-beam computed tomography (CBCT) are helpful for locating supernumerary teeth, but the images cannot be transferred to the operation. To design a novel surgical guide plate for intraoperative navigation, we transfer the patient's oral CBCT and gypsum model scan data to a computer for analysis. In our study, we evaluate the efficiency and safety of a novel surgical guide plate for the extraction of deeply impacted supernumerary teeth (DIMSNT) in the anterior maxilla. Methods: Forty patients treated at the Department of School & Hospital of Stomatology, Wenzhou Medical University from March 2019 to December 2020 with DIMSNT (type II/III according to Liu et al.) in the anterior maxilla were randomly divided into 2 groups (20 patients for each group) for the extraction. For group I, a novel surgical guide was selected using CBCT and gypsum model scan. In contrast, for group II who underwent freehand surgery, only the CBCT data was used. The evaluation of operation time, complications, satisfaction score, and the number of cases that underwent extraction immediately after removing the bone were performed to assess the efficiency and safety of this novel surgical plate. Results: All patients completed the surgery successfully. The guides for group I had a good application effect. Group I's operation time (23.35±5.39 min) was shorter than group II (29.60±9.76 min) (P=0.0194). The average pain degree of group I (1.8±1.08) was significantly less than group II (2.82±1.68) (P<0.05). The average swelling score of group I (34) was significantly less than group II (44.7). Patient satisfaction was significantly higher in group I (8.95±1.05) than in group II (7.90±1.51) (P=0.0152). Conclusions: The novel surgical guide assisted with DIMSNT extraction have been effective in improving the quality of the surgery, patient satisfaction, and reduce its difficulty and duration. We can construct a surgical guide plate to guide the incision and osteotomy in DIMSNT surgery through the data analysis of DIMSNT on computer, which has a broad application prospect for clinical use. Trial registration: Chinese Clinical Trial Registry ChiCTR2100054523.

Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography.

N-terminal glutamate (E) cyclization to form pyroglutamate (pE) generates charge heterogeneities for mAbs and proteins. Thus far, pE formation rate in lyophilized formulation as compared to in liquid formulation has not been reported. Impact of pE on antibody biological activity has only been predicted or assessed using stressed samples that may contain other confounding degradations besides pE. Additionally, application of hydrophobic interaction chromatography (HIC) to separate pE has not been reported. In our study, N-terminal E cyclization was identified as the major degradation pathway in lyophilized formulation at elevated temperature for both monoclonal antibody (mAb-A) and IgG-like bispecific antibody (bsAb-A). pE was enriched in salt-gradient ion exchange chromatography (IEC) as pre-peak and in HIC as post-peak for both mAb-A and bsAb-A. Structure-function studies with pE-enriched IEC and HIC fractions confirmed that pE did not affect binding activities for mAb-A and bsAb-A. In vitro incubation of bsAb-A in serum and PBS revealed that the serum matrix may play a role in pE conversion in human serum, in contrast to the chemical reaction mechanism reported. These techniques can help in characterization of N-terminal E-to-pE cyclization and quality attribute severity assessment during therapeutic protein product development.

The impact of land use and rainfall patterns on the soil loss of the hillslope.

In order to discuss the effect of rainfall patterns and land use types on soil erosion, the experiment is carried out under natural rainfall events on different kinds of runoff plots in Zhangjiachong watershed. Based on the observed data of 44 individual rainfall events including moderate, heavy and storm rainfall, the differences of erosion modulus among hedgerows plots, terrace plots, and slope plots under different rainfall patterns are analyzed. And the effects of hedgerow and terrace patterns on control of soil loss are revealed by RUSLE. Wilcoxon signed rank test is applied to analyze the significant difference of erosion modulus in different plots and the coefficient of variation is used to compare the characteristics of erosion modulus under different rainfall patterns. The results show that the soil erosion modulus of earth banked terrace has the highest value and the lowest soil erosion modulus occurs in the slope land with hedgerows. The coefficients of variation for soil erosion modulus under heavy and storm rainfall are larger than that of moderate rainfall. Hedgerow pattern can effectively control soil erosion under moderate and heavy rainfall while the effect of hedgerow is considerably weakened under storm rainfall. Earth banked terraces own the highest erosion modulus followed by slope land and stone dike terraces.

Efficaciousness of dexmedetomidine in children undergoing cleft lip and palate repair: a systematic review and meta-analysis.

OBJECTIVE: To systematically assess the efficacy and safety of dexmedetomidine as an anaesthesia adjuvant for cleft lip and palate (CLP) repair in children. DESIGN: Systematic review and meta-analysis. DATA SOURCES: PubMed, Embase, Cochrane, China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (VIP) and Wanfang (up to October 2020). Studies in languages other than English and Chinese were excluded. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Randomised controlled trials (RCTs) evaluating the impact of dexmedetomidine on emergence agitation (EA), the need for postoperative rescue analgesics, postoperative nausea and vomiting (PONV), and other adverse events in paediatric patients during CLP repair. DATA EXTRACTION AND SYNTHESIS: The quality of evidence was assessed by using the Cochrane Review Methods and the Grading of Recommendations Assessment, Development and Evaluation approach. Data were screened, extracted and assessed by two independent authors. Outcomes were reported as a risk ratio (RR) with a 95% CI. A random-effect model was used when heterogeneity was detected. RESULTS: Thirteen studies including 1040 children met the inclusion criteria. The incidence of EA was significantly decreased in the dexmedetomidine group (RR, 0.19; 95% CI 0.10 to 0.36; p<0.00001; I2=56%) as compared with the control group. Paediatric patients receiving dexmedetomidine had lower postoperative analgesic requirements (RR, 0.27; 95% CI 0.10 to 0.73; p=0.01; I2=84%) and a lower incidence of respiratory adverse events (RR, 0.49; 95% CI 0.31 to 0.78; p=0.003; I2=0%). There were no significant differences in the risk of PONV and cardiovascular adverse events. CONCLUSIONS: There was a lack of high-quality studies in this field. Perioperative administration of dexmedetomidine reduced the need for postoperative rescue analgesics and the incidence of EA in children without side effects undergoing CLP repair. However, further verification with larger samples and higher-quality RCTs is needed.

Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1.

T-cell-mediated immunotherapy has generated much excitement after the success of therapeutic biologics targeting immune checkpoint molecules. Bispecific antibodies (BsAbs) that recognize two antigen targets are a fast-growing class of biologics offering promising clinical benefits for cancer immunotherapy. Due to the complexity of the molecule structure and the potential mechanism of action (MOA) that involves more than one signaling pathway, it is critical to develop appropriate bioassays for measuring potency and characterizing the biological properties of BsAbs. Here, we present a dual target, cell-based reporter bioassay for a BsAb that binds human CTLA-4 and PD-1 and targets two subsequent signaling pathways that negatively regulate T-cell activation. This reporter bioassay is capable of measuring the potency of both antigen target arms in one assay, which would not be achievable using two single target bioassays. This dual target reporter bioassay demonstrates good performance characteristics suitable for lot release, stability testing, critical quality attribute assessment, and biological properties characterization of the CTLA-4/PD-1 BsAb. Furthermore, this assay can capture the synergistic effect of anti-CTLA-4 and anti-PD-1 activity of the BsAb. Compared to single target assays, this dual target bioassay could better reflect the potential MOA of BsAbs and could be used for evaluation of other bispecific biologics, as well as antibody combination therapies.

Enhanced remediation efficiency of Cr(VI)-contaminated heterogeneous aquifers: Improved sweeping efficiency using shear-thinning fluids.

Low permeability zones (LPZs) are typically bypassed when remedial reagents are injected into heterogeneous aquifers, which hinders the in situ remediation. Although shear-thinning polymers have emerged as promising tools to meet this challenge, their applicability in complex remedial systems remains unconfirmed. We investigated the sweeping efficiencies of calcium polysulfide (CPS) into Cr(VI)-contaminated LPZs using xanthan gum (XG) as the model shear-thinning polymer. Firstly, the compatibility of XG-CPS fluids and their reduction capacities toward Cr(VI) were demonstrated based on batch experiments. The removal rates of Cr(VI) exceeded 85% in the presence of 250-2000 mg/L of XG. Besides, XG-CPS fluids exhibited a greater impact on the permeability decrease of transmissive zones than that of LPZs as confirmed by sand column experiments. Furthermore, the sweeping efficiencies in LPZs during XG-CPS flooding were investigated by multiple sand tank experiments. The sweeping rate in LPZs (rs) in Cr(VI)-contaminated aquifer (1.68 × 10-3/min) was found to be approximately 11% higher than that of uncontaminated system, and two possible reasons behind this phenomenon were proposed. The spatial distribution profiles of Cr under different XG-CPS flooding conditions were depicted based on 20 representative samples. The results indicated that all Cr(VI) in LPZs can be effectively removed either by displacement or immobilization as Cr(III). The percentages of displaced Cr(VI) and immobilized Cr(III) were calculated to be 65%-75% and 25-35%, respectively. This work demonstrates the applicability of XG-CPS fluids as remedial materials for Cr(VI)-contaminated heterogeneous aquifers and provides novel insights into the role of Cr(VI) in in situ remediation using shear-thinning polymers.

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation.

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2.

Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 1011 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping.

mAbs undergo several post-translational modifications, including the formation of succinimide from the deamidation of asparagine or the isomerization of aspartic acid. Because of the potential impact of succinimide formation on the biological activity of mAbs, detection and quantification of this species is a key area of interest for the pharmaceutical industry. However, studies assessing succinimide stability have been limited, and methods developed to monitor succinimide are either product specific or not robust. Here, we report the development of a platform low-pH peptide-mapping method using a combination of low-pH-resistant Lys-C and modified trypsin to maintain succinimide stability, eliminate deamidation assay artifact, and achieve efficient mAb digestion equivalent to conventional tryptic peptide-mapping method under alkaline condition. Using this method, succinimide stability in serum was accurately assessed in vitro study and the half-life was determined to be 1.5 days. With potential patient exposure to succinimide intermediate, a reliable method was developed to measure site-specific deamidation and succinimide intermediate. Coupled with a single quadrupole mass detector, our method was automated from digestion to data processing and applicable in a good manufacturing practice environment. The method was fully qualified to demonstrate accuracy, precision, linearity, and robustness.

Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis.

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.

The influence of ACYP2 polymorphisms on gastrointestinal cancer susceptibility in the Chinese Han population.

BACKGROUND: Gastrointestinal cancer (GI cancer) is a type of cancer that has a high death rate. It has been reported that ACYP2 gene was associated with the development of gastric cancer and colorectal cancer, but it is not clear that the relationship between ACYP2 gene and GI cancer in Chinese Han population. This study aimed to investigate the association between polymorphisms of ACYP2 and GI cancer in the Chinese Han population. METHODS: We used Agena MassARRAY to determine the genotypes of 1,160 GI cancer patients and 495 healthy controls. The correlation between ACYP2 variants and GI cancer risk was examined by logistic regression analysis. RESULTS: We identified that rs6713088 (OR = 1.17, 95% CI: 1.00-1.36, p = 0.047), rs843711 (OR = 1.17, 95 CI: 1.01-1.36, p = 0.035), and rs11896604 (OR = 1.20, 95% CI: 1.00-1.45, p = 0.048) were correlated with an increased risk of GI cancer under allele model. Rs11125529 under the recessive model (OR = 2.05, 95% CI: 1.00-4.23, p = 0.038), rs843711 in recessive model (OR = 1.37, 95% CI: 1.04-1.82, p = 0.026), and rs11896604 under log-additive model (OR = 1.23, 95% CI: 1.01-1.51, p = 0.042) were associated with an increased risk of GI cancer. CONCLUSION: Our study suggested that polymorphisms of ACYP2 gene might be associated with susceptibility to GI cancer.

A method to directly analyze free-drug-related species in antibody-drug conjugates without sample preparation.

Residual free-drug-related species that are present in antibody-drug conjugates (ADC) are a potential safety risk to patients and are therefore categorized as a critical quality attribute that must be strictly monitored and controlled. Among the many analytical methods developed for free-drug analysis, reversed-phase liquid chromatography (RP-LC) is the most common approach. Conventional RP-LC methods for free-drug analysis, however, involve labor-intensive sample preparation. Here we present a new RP-LC method to directly analyze free-drug-related species in an ADC sample without the need for sample preparation. In our work, free-drug-related species were very well separated from ADC peaks in the chromatography gradient. Typical performance issues observed in conventional RP-LC, such as column fouling, detection interference, and carryover, were not observed or were negligible with this new method. Three options were evaluated for free-drug quantitation: Strohl (2017) [1] use of an external free drug calibration curve for determination of absolute concentration; Perez et al. (2014) [2] calculation of relative percentage based on peak area ratio between free drug and ADC at a characteristic wavelength unique for drug payload; and (Beck et al., 2017) [3] calculation of relative percentage based on peak area ratio between free drug and corrected ADC peak area (at any wavelength). The method with calibration curve provides the highest sensitivity, the best accuracy and precision for determination of free drug present in the ADC. However, the second and third options were simpler because they eliminated the need for an external calibration curve, making them worth exploring. Due to its simplicity and compatibility with mass spectrometry, the new method is also a good choice for direct analysis of stability samples.