Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.

Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.

Does competition support integrated care to improve quality?

Introduction: This work investigates the compatibility of integrated care and competition in China and analyses the impact of integrated care on regional care quality (DeptQ) within a competitive framework. Method: The study was built on multivariate correspondence analysis and a two-way fixed-effects model. The data were collected from Xiamen's Big Data Application Open Platform and represent nine specialised departments that regularly performed inter-institutional referrals between 2016 and 2019. Results: First, care quality for referred patients (ReferQ) and the relative scale of referred patients (ReferScale) and competition have an antagonistic but not completely mutually exclusive relationship. Second, ReferQ and competition both have a significant effect on DeptQ, but only when competition is weak can ReferQ and competition act synergistically on DeptQ. When competition is fierce, competition will weaken the impact of ReferQ on DeptQ. Conclusion: Changes in the intensity of integrated care and competition ultimately affect care quality.

Multilevel synergically controlling wavefront correction of a high-power slab laser system.

We present a multilevel synergically controlling wavefront correction method that can apply in a slab laser system. To fully utilize the response frequency and the stroke of actuators of the single deformable mirror (DM), we design a set of multilevel wavefront correction devices to reduce the root-mean square of wavefront aberration before the DM. As the wavefront of slab geometry solid-state lasers mainly consists of fourth and longitudinally distributed aberration, such as 5th, 9th, and 14th orders of Legendre polynomials. We design a precompensating level of the aberration with a slow-drift mirror, fast-steer mirror, one-dimensional adjustable slab-aberration compensator, and beam-shaping system to reduce these orders of wavefront aberration with low spatial resolution and large stroke. As the controlling bandwidth of different devices is diverse, the coupling oscillation between the precompensating level and adaptive optics (AO) level occurs, then we develop the multilevel synergically control to address the coupling. With the precompensating level, the experimental result shows the residual wavefront aberration of the slab laser is compensated well by the AO level effectively within the compensating capability. We clean up a 9.8 kW slab laser system with the beam quality ß of far-field focus spots improved from 17.71 to 2.24 times the diffraction limit.

[Spatial distribution and the influencing factors of organic carbon of biological crusts on regional scale in Mu Us sandy land, China]. / æ¯›ä¹Œç´ æ²™åœ°åŒºåŸŸå°ºåº¦ç”Ÿç‰©ç»“çš®æœ‰æœºç¢³.

As an important soil cover in deserts, biological crusts play a central role in ecosystem function such as nutrient cycling, nitrogen fixation, and carbon sequestration. Many biological crust organisms could fix CO2 through photosynthesis to improve soil organic carbon content. There is a knowledge gap in the origin of soil organic carbon (SOC) from biological crusts on a regional level, which restricts the prediction of soil carbon pool. Based on 45 plots in the Mu Us sandy land (42200 km2), we measured the SOC content and soil organic carbon density (SOCD) of two types of typical biological crusts (moss crusts, algal crusts) and their underlying soils, and analyzed together with the climate data, soil and vegetation factors to investigate the spatial distribution characteristics and controlling factors of organic carbon of biological crusts at the regional scale. The results showed that: 1) biological crusts significantly increased SOC and SOCD compared with bare ground. Moss crusts and the underlying SOC (4.93 g·kg-1) and SOCD (0.41 kg·m-2) were higher than those of algal crusts (1.89 g·kg-1, 0.18 kg·m-2). 2) On the regional scale, the SOC and SOCD of biological crusts had clear spatial distribution characteristics, demonstrating a banded distribution and block mosaic from northeast to central and west to southeast. 3) The SOC and SOCD of biological crusts and their underlying soils were mainly affected by climate, soil and vegetation conditions, while the main controlling factors depended on the types of biological crusts. The SOC and SOCD of moss crust were controlled by annual maximum temperature and potential evapotranspiration, whereas those of algal crusts were controlled by water vapor pressure.

[Effects of biological crusts on soil organic carbon in sandlands under a precipitation gradient]. / 不同降水条件下生物结皮覆盖对沙地土壤有机碳的影响.

Biological crusts (Biocrusts) are important surface active coverings in arid and semi-arid regions, which affect the content of soil organic carbon (SOC), SOC labile fractions and stability of SOC through photosynthetic carbon fixation. At present, studies on the variation characteristics of SOC, SOC labile fractions and the stability of SOC in biocrusts are rather limited. In this study, two types of typical biocrusts (moss crusts and algae crusts) were selected along a precipitation gradient from northwest to southeast in the Mu Us Sandland (straight line distance 188 km) by measuring soil organic carbon (SOC), soil microbial biomass carbon (MBC), water soluble carbon (DOC), particulate carbon (POC), easily oxidizable carbon (ROC). We aimed to explore the effects of biocrusts on the stability of SOC and carbon decomposition across the precipitation gradient. Results showed that:1) Two types of biocrusts significantly increased the contents of SOC, MBC, DOC, POC, ROC and stability of SOC. Moss crusts increased SOC contents by 1.6 to 2.6 times as that of algae crusts. 2) The lowest SOC contents of the two types of biocrusts were 6.43 g·kg-1 and 14.50 g·kg-1 respectively, which showed an increasing trend with increasing precipitation along the gradient. 3) With the increases of precipitation, the decomposition time of moss litters gradually decreased. The decomposition coefficient of moss litters during the study period (From July to Feb-ruary of the next year) ranged from 0.010 to 0.014, which was significantly lower than that of vascular plants. The carbon release of moss litters from northwest to southeast was 8.09, 10.89, 12.88 g·kg-1, respectively. 4) Results of canonical correspondence analysis showed that water vapor partial pressure, actual evapotranspiration, annual average temperature, subsurface short-wave radiation, potential evapotranspiration and vapor pressure difference were the key climate factors affecting the content of SOC and its active components. Silt content was the main soil factor affecting the content of SOC and its active components.

G protein-coupled estrogen receptor promotes acrosome reaction via regulation of Ca2+ signaling in mouse sperm†.

G protein-coupled estrogen receptor (GPER), a seven-transmembrane G protein-coupled receptor, mediates the rapid pre-genomic signaling actions of estrogen and derivatives thereof. The expression of GPER is extensive in mammal male reproductive system. However, the functional role of GPER in mouse sperm has not yet been well recognized. This study revealed that GPER was expressed at the acrosome and the mid-flagellum of the mouse sperm. The endogenous GPER ligand 17ß-estradiol and the selective GPER agonist G1 increased intracellular Ca2+ concentration ([Ca2+]i) in mouse sperm, which could be abolished by G15, an antagonist of GPER. In addition, the G1-stimulated Ca2+ response was attenuated by interference with the phospholipase C (PLC) signaling pathways or by blocking the cation channel of sperm (CatSper). Chlortetracycline staining assay showed that the activation of GPER increased the incidence of acrosome-reacted sperm. Conclusively, GPER was located at the acrosome and mid-flagellum of the mouse sperm. Activation of GPER triggered the elevation of [Ca2+]i through PLC-dependent Ca2+ mobilization and CatSper-mediated Ca2+ influx, which promoted the acrosome reaction of mouse sperm.

Primary aberration optimization for double-plane symmetric beam shaping systems using a pair of curved reference surfaces.

The geometric aberration of centered refracting double-plane symmetric optical systems (DPSOS) is investigated. For DPSOS with different defocus values in the tangential plane and the sagittal plane (astigmatic wavefront), a pair of curved reference surfaces which vanishes the quadratic terms of the optical path difference (OPD) between a general ray and a reference ray are deduced. With the curved reference surfaces, the primary (fourth-order) wave aberration function for DPSOS is calculated and analyzed, which can be used for beam shaping designs with astigmatic input wavefront, such as slab lasers and semiconductor lasers. Further, the proposed curved reference surfaces can be applied to analyze the aberrations of general DPSOS.

Compact integrated aberration-compensating module for a 9 kW Nd:YAG slab laser based on an off-axis stable-unstable resonator.

An integrated aberration-compensating module (IACM), consisting mainly of an adjustable slab-aberration compensator, a one-dimensional Shack-Hartmann wavefront sensor, and a data processor, which meet the urgent requirements of correcting the specific wavefront aberrations of a slab laser based on an off-axis stable-unstable resonator, is designed and experimentally demonstrated. Benefits include compactness, robustness, simplicity, automation, and cost-effectiveness. The particular wavefront aberrations of the 9 kW level quasi-continuous-wave Nd:YAG slab laser, which have characteristics of asymmetry, large amplitude and gradient, high spatial frequency, and low temporal frequency, were measured and theoretically analyzed. In the experiment, the wavefront aberrations of the slab laser were corrected by the IACM. At the average output power of 9 kW, the diffraction-limited factor ß was improved from 20.3 times diffraction limit (DL) to 3.6 times DL. The peak-to-valley and root-mean-square values of aberrations were reduced from 9.6 to 0.85 µm and from 2.86 to 0.18 µm within five iterations of the IACM, respectively. Moreover, The IACM is capable of maintaining the compensating surface figure after power-off.

Simplified analytical method for an anamorphic refractive shaping system of laser beams with a large aspect ratio.

For reshaping aperture size and correcting low-order aberration of laser beams with large aspect ratios, a simplified analytical method is proposed to design an anamorphic refractive shaping system, which is composed of double-plane symmetric lenses. The simplified method enables performing a global study of aberrations via calculating the analytical primary wave aberration function under paraxial approximation. The aberration balance is analyzed with a three-lens laser collimating system and a compact four-lens laser expanding system. Lens bending and conic surfaces are introduced to decrease ray errors. Through the simplified analytical method, anamorphic refractive shaping systems for laser beams with large aspect ratios can be adequately analyzed and conveniently designed.

A Highly Sensitive LC-MS/MS Method for Targeted Quantitation of Lipase Host Cell Proteins in Biotherapeutics.

Identification and accurate quantitation of host cell proteins (HCPs) in biotherapeutic drugs has become increasingly important due to the negative impact of certain HCPs on the safety, stability, and other product quality of biotherapeutics. Recently, several lipase HCPs have been identified to potentially cause the enzymatic degradation of polysorbate, a widely used excipient in the formulation of biotherapeutics, which can severely impact the stability and product quality of drug products. In this study, we identified three lipase HCPs that were frequently detected in Chinese hamster ovary (CHO) cell cultures using shotgun proteomics, including phospholipase B-like 2 (PLBL2), lipoprotein lipase (LPL), and lysosomal acid lipase (LIPA). A targeted quantitation method for these three lipase HCPs was developed utilizing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with high-resolution multiple-reaction-monitoring (MRMhr) quantitation. The method demonstrated good sensitivity with low limit of quantitation (LLOQ) around 1 ng/mL, and linear dynamic range of three orders of magnitude for the three lipase HCPs. It has been applied for the characterization of process intermediates from various in-house monoclonal antibody (mAb) production. In addition, the method has also been used to evaluate the robustness of clearance for one of the lipase HCPs, PLBL2, under different column purification process conditions.

Excellent performance of a cryogenic Nd:YAlO3 laser with low wavefront distortion based on zero thermal expansion.

High-power solid-state lasers with good beam quality are attracting great attention on account of their important applications in industry and military. However, the thermal effects generated in the laser host materials seriously limit power scaling and degrade the beam quality. Thermal lensing and thermally induced wavefront deformation are the main causes of the beam quality deterioration. Here we investigate the performance of a zero thermal expansion (ZTE) solid-state laser gain material. In a proof-of-principle experiment, an ${a}$-cut rod ${\rm Nd}\!:\!{{\rm YAlO}_3}$ (Nd:YAP) perovskite crystal is chosen to be the gain medium for ZTE around 180 K. The laser performance spanning the temperature range from 80 to 290 K is studied. The maximum output power and minimum threshold pump power were obtained at a temperature of 180 K. Moreover, the measured thermal focal power and peak-to-valley value of the wavefront distortion also reach a minimum at this temperature, an additional benefit from the crystal's ZTE coefficient. We envisage that these results will open a new route towards the development of high-power and high-beam-quality lasers through the use of ZTE gain materials.

Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development.

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.

Brain magnetic resonance imaging findings and radiologic review of maple syrup urine disease: Report of three cases.

BACKGROUND: Maple syrup urine disease (MSUD) is a rare autosomal-recessive disorder that affects branched-chain amino acid (BCAA) metabolism and is named after the distinctive sweet odor of affected infants' urine. This disease is characterized by the accumulation of BCAAs and corresponding branched-chain ketoacids of leucine, isoleucine, and valine in the plasma, urine, and cerebrospinal fluid. However, the mechanisms of MSUD-induced brain damage remain poorly defined. The accumulation of BCAAs in the brain inhibits the activity of pyruvate dehydrogenase and α-ketoglutarate, disrupting the citric acid cycle and consequently impacting the synthesis of amino acids, causing cerebral edema and abnormal myelination. CASE SUMMARY: We report three neonates admitted to our hospital with the classic subtype of MSUD. All three patients, with a transient normal period, presented with poor feeding, vomiting, poor weight gain, and increasing lethargy after birth. Laboratory testing revealed metabolic acidosis. The serum tandem mass spectrometry amino acid profile showed elevated plasma levels of BCAAs (leucine, isoleucine, and valine). Brain magnetic resonance imaging (MRI) presented abnormal signals mainly involving the globus pallidus, thalamus, internal capsule, brainstem, and cerebellar white matter, which represent the typical myelinated areas in normal full-term neonates. CONCLUSION: In our patients, MRI showed typical features, in concordance with the available literature. Early detection and timely treatment are very helpful for the prognosis of MSUD patients. Therefore, we discuss the neuroimaging features of MSUD to enhance the knowledge of pediatricians about this disease.

Imaging manifestations of congenital mesoblastic nephroma.

OBJECTIVE: Congenital mesoblastic nephroma (CMN) is a rare renal tumor mainly observed in infants and young children. This study aims to analyze the imaging manifestations of CMN to improve the understanding of the disease. METHODS: The imaging manifestations and clinical records of all pediatric patients with CMN admitted to our hospital over the last 7 years were retrospectively analyzed. The diagnosis of CMN was confirmed by postoperative pathology. All patients underwent computed tomography (CT) scans; 2 patients additionally underwent magnetic resonance imaging (MRI) scans (including one prenatal MRI scan). RESULTS: We evaluated 10 pediatric patients (6 males and 4 females) aged 7 days to 12 months (median age: 4 months) with CMN located on the left kidney in six cases and the right kidney in four cases. The CT imaging manifested as solid lesions (5 cases), solid-cystic lesions with solid predominance (4 cases), or solid-multicystic lesions with cystic predominance (1 case). Enhanced CT showed moderately and heterogeneously enhanced solid component and intracystic septations at the corticomedullary phase that were further enhanced at the nephrographic phase, although their CT values were still lower than those of the renal parenchyma. The "double-layer sign" were seen in 4 cases of classic type of CMN, and the "intratumor pelvis sign" were seen in 9 cases that include 5 classic, 3 cellular and 1 mixed type of CMN. In the 2 patients who underwent MRI, the scans showed solitary masses. The lesions had hypointense signals on the T1WI sequence and isointensity or slightly lower-intensity signals than the surrounding renal parenchyma on the fluid-sensitive sequences, whereas the lesions showed hyperintense signals on the diffusion-weighted imaging (DWI) sequence. CONCLUSIONS: The imaging manifestations of CMN are closely correlated with the pathological subtype and have certain characteristics. The "double-layer sign" was seen with most classic type CMN, and "intratumor pelvis sign" was seen in 90% cases.

Performances of Prognostic Models in Stratifying Patients with Advanced Gastric Cancer Receiving First-line Chemotherapy: a Validation Study in a Chinese Cohort

Purpose@#While several prognostic models for the stratification of death risk have been developed for patients with advanced gastric cancer receiving first-line chemotherapy, they have seldom been tested in the Chinese population. This study investigated the performance of these models and identified the optimal tools for Chinese patients. @*Materials and Methods@#Patients diagnosed with metastatic or recurrent gastric adenocarcinoma who received first-line chemotherapy were eligible for inclusion in the validation cohort. Their clinical data and survival outcomes were retrieved and documented. Time-dependent receiver operating characteristic (ROC) and calibration curves were used to evaluate the predictive ability of the models. Kaplan-Meier curves were plotted for patients in different risk groups divided by 7 published stratification tools. Log-rank tests with pairwise comparisons were used to compare survival differences. @*Results@#The analysis included a total of 346 patients with metastatic or recurrent disease.The median overall survival time was 11.9 months. The patients were different into different risk groups according to the prognostic stratification models, which showed variability in distinguishing mortality risk in these patients. The model proposed by Kim et al. showed relative higher predicting abilities compared to the other models, with the highest χ 2 (25.8) value in log-rank tests across subgroups, and areas under the curve values at 6, 12, and 24 months of 0.65 (95% confidence interval [CI]: 0.59–0.72), 0.60 (0.54–0.65), and 0.63 (0.56–0.69), respectively. @*Conclusions@#Among existing prognostic tools, the models constructed by Kim et al., which incorporated performance status score, neutrophil-to-lymphocyte ratio, alkaline phosphatase, albumin, and tumor differentiation, were more effective in stratifying Chinese patients with gastric cancer receiving first-line chemotherapy.

The Value of Enhanced MR Radiomics in Estimating the IDH1 Genotype in High-Grade Gliomas.

BACKGROUND: The prognosis of IDH1-mutant glioma is significantly better than that of wild-type glioma, and the preoperative identification of IDH mutations in glioma is essential for the formulation of surgical procedures and prognostic assessment. PURPOSE: To explore the value of a radiomic model based on preoperative-enhanced MR images in the assessment of the IDH1 genotype in high-grade glioma. MATERIALS AND METHODS: A retrospective analysis was performed on 182 patients with high-grade glioma confirmed by surgical pathology between December 2012 and January 2019 in our hospital with complete preoperative brain-enhanced MR images, including 79 patients with an IDH1 mutation (45 patients with WHO grade III and 34 patients with WHO grade IV) and 103 patients with wild-type IDH1 (33 patients with WHO grade III and 70 patients with WHO grade IV). Patients were divided into a primary dataset and a validation dataset at a ratio of 7 : 3 using a stratified random sampling; radiomic features were extracted using A.K. (Analysis Kit, GE Healthcare) software and were initially reduced using the Kruskal-Wallis and Spearman analyses. Lasso was finally conducted to obtain the optimized subset of the feature to build the radiomic model, and the model was then tested with cross-validation. ROC (receiver operating characteristic curve) analysis was performed to evaluate the performance of the model. RESULTS: The radiomic model showed good discrimination in both the primary dataset (AUC = 0.87, 95% CI: 0.754 to 0.855, ACC = 0.798, sensitivity = 85.5%, specificity = 75.4%, positive predictive value = 0.734, and negative predictive value = 0.867) and the validation dataset (AUC = 0.86, 95% CI: 0.690 to 0.913, ACC = 0.789, sensitivity = 91.3%, specificity = 69.0%, positive predictive value = 0.700, and negative predictive value = 0.909). CONCLUSION: The radiomic model, based on the preoperative-enhanced MR, can effectively predict the IDH1 genotype in high-grade glioma.

Multimodal MR Features of 8 Cases of Epithelioid Glioblastoma.

PURPOSE: The MRI features of epithelioid glioblastoma (eGBM) were analyzed. The apparent diffusion coefficient (ADC), MR perfusion-weighted imaging (PWI), and magnetic resonance spectroscopy (MRS) findings were quantitatively analyzed. METHODS: The MRI images of 8 cases of eGBM were analyzed retrospectively. The location and edge, signal, peritumoral edema, adjacent meningeal invasion, and enhancement of the lesions were observed. The ADC value, relative cerebral blood volume (rCBV), relative cerebral blood flow (rCBF), and N-acetylaspartate/acetylcholine (NAA/Cho) value were analyzed. RESULTS: Among the 8 patients, the tumors were mainly located in the temporal lobe (n = 3), frontal lobe (n = 3), and parietal lobe (n = 2). The lesion boundary was clear in 6 cases and unclear in 2. The lesions were superficial in 5 cases and in the deep white matter in 3. Internal hemorrhage was observed in 4 cases. There was cystic necrosis in 7 cases, and only 1 case was solid without cystic necrosis. There was no edema around the lesion in 1 case, severe edema in 5, and moderate edema in 2. In 4 cases, the adjacent meninges were involved, and in 1 case, the ependyma was involved. Two patients developed leptomeningeal metastasis within 2 months after the operation. The average ADC value of the tumor parenchyma among all 8 patients was7.15 × 10-4 mm2/s,which was 17.6% lower than that of the contralateral side. The Cho/NAA metabolite ratio was 5.27 and 0.81 in the lesions of 2 patients. The rCBV was 3.51 ml/100 g and 3.32 ml/100 g of lesions in 2 patients; these values were 36% and 29% higher, respectively, than those of the contralateral side. The rCBF was 31.5 ml/100 g/min and 82.1 ml/100 g/min of lesions in two patients; these values were 49% and 203% higher, respectively, than those of the contralateral side. CONCLUSION: eGBM characteristics include a superficial location, easy cyst degeneration, easy necrosis and hemorrhage, and clear boundaries. It easily invades adjacent meninges and shows cerebrospinal fluid dissemination and metastasis. Combining new MR techniques, such as ADC values, PWI, and MRS, could be helpful for improving diagnostic accuracy.

The Value of Corpus Callosum Measurement in the Diagnosis of Cerebral Atrophy.

OBJECTIVE: The study aimed to investigate the relationship between the corpus callosum area (CCa) and the degree of cerebral atrophy in patients with cerebral atrophy. METHODS: 119 patients with brain atrophy were grouped according to the degree of brain atrophy. Median sagittal CCa and intracranial area (ICa) were measured, and the ratio of corpus callosum to the intracranial area (CCa-ICa ratio) was calculated. The data were analyzed using ANOVA. RESULTS: CCa significantly reduced in patients with cerebral atrophy, and the degree of cerebral atrophy was found to be positively correlated with the degree of reduction in the CCa. CONCLUSION: The reduction in the CCa and the CCa-ICa ratio in the median sagittal can be used as a reference indicator for the diagnosis and grading of brain atrophy in clinical practice.

Sequence Variant and Posttranslational Modification Analysis During Cell Line Selection via High-Throughput Peptide Mapping.

Selection of high-producing lead and backup cell lines with high-fidelity primary structure is a major goal of cell line development of protein therapeutics. Conventional techniques for sequence variant analysis, such as mass spectrometry (MS) and next-generation sequencing (NGS) have limitations on the sample number and turnaround time, thus often are only applied at the final stages of development, where an undesired lead or backup clone could cause a significant delay in project timeline. Here we presented a high-throughput (HT) peptide mapping workflow which can be applied at early stages of cell line selection for testing of a batch of 30-40 clones within 2-week turnaround while reporting valuable information on sequence variants and posttranslational modifications (PTMs). The successful application of this workflow was demonstrated for two mAb programs. Multiple clones were removed from a total of 33 mAb-1 clones using various criteria: nine clones contained at least one >1% upregulated unknown peptide ions, 11 clones contained at least eight >0.1% upregulated unknowns, and six clones contained upregulated critical PTMs. For mAb-2, light chain (LC) sequence extension of approximately 30 amino acids were detected in 6 out of 36 clones at levels up to 11%. Besides, a Q to H mutation at ~30% was detected in the heavy chain (HC) of a single clone. Q to H mutation has mass change of 9.00 Da and failed to be detected by intact mass analysis. Rapid PTM quantitation also facilitated the selection of clones with desirable quality attributes, such as N-glycan profile. Hence, we demonstrated a risk-reducing strategy where abnormal clones could be detected at earlier stages of cell line selection, which should result in reduced and more predictable timeline of cell line development.