Understanding and controlling the molecular mechanisms of protein aggregation in mAb therapeutics.

In antibody development and manufacturing, protein aggregation is a common challenge that can lead to serious efficacy and safety issues. To mitigate this problem, it is important to investigate its molecular origins. This review discusses (1) our current molecular understanding and theoretical models of antibody aggregation, (2) how various stress conditions related to antibody upstream and downstream bioprocesses can trigger aggregation, and (3) current mitigation strategies employed towards inhibiting aggregation. We discuss the relevance of the aggregation phenomenon in the context of novel antibody modalities and highlight how in silico approaches can be exploited to mitigate it.

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion.

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.

Semi-Automated Glycoproteomic Data Analysis of LC-MS Data Using GlycopeptideGraphMS in Process Development of Monoclonal Antibody Biologics.

The glycosylation of antibody-based proteins is vital in translating the right therapeutic outcomes of the patient. Despite this, significant infrastructure is required to analyse biologic glycosylation in various unit operations from biologic development, process development to QA/QC in bio-manufacturing. Simplified mass spectrometers offer ease of operation as well as the portability of method development across various operations. Furthermore, data analysis would need to have a degree of automation to relay information back to the manufacturing line. We set out to investigate the applicability of using a semiautomated data analysis workflow to investigate glycosylation in different biologic development test cases. The workflow involves data acquisition using a BioAccord LC-MS system with a data-analytical tool called GlycopeptideGraphMS along with Progenesis QI to semi-automate glycoproteomic characterisation and quantitation with a LC-MS1 dataset of a glycopeptides and peptides. Data analysis which involved identifying glycopeptides and their quantitative glycosylation was performed in 30 min with minimal user intervention. To demonstrate the effectiveness of the antibody and biologic glycopeptide assignment in various scenarios akin to biologic development activities, we demonstrate the effectiveness in the filtering of IgG1 and IgG2 subclasses from human serum IgG as well as innovator drugs trastuzumab and adalimumab and glycoforms by virtue of their glycosylation pattern. We demonstrate a high correlation between conventional released glycan analysis with fluorescent tagging and glycopeptide assignment derived from GraphMS. GraphMS workflow was then used to monitor the glycoform of our in-house trastuzumab biosimilar produced in fed-batch cultures. The demonstrated utility of GraphMS to semi-automate quantitation and qualitative identification of glycopeptides proves to be an easy data analysis method that can complement emerging multi-attribute monitoring (MAM) analytical toolsets in bioprocess environments.

Utility of Ion-Mobility Spectrometry for Deducing Branching of Multiply Charged Glycans and Glycopeptides in a High-Throughput Positive ion LC-FLR-IMS-MS Workflow.

High-throughput glycan analysis has become an important part of biopharmaceutical production and quality control. However, it is still a significant challenge in the field of glycomics to easily deduce isomeric glycan structures, especially in a high-throughput manner. Ion mobility spectrometry (IMS) is an excellent tool for differentiating isomeric glycan structures. However, demonstrations of the utility of IMS in high-throughput workflows such as liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) workflows have been limited with only a small amount of collision cross section (CCS) data available. In particular, IMS data of glycan fragments obtained in positive ion mode are limited in comparison to those obtained in negative ion mode despite positive ion mode being widely used for glycomics. Here, we describe IMS TWCCSN2 data obtained from a high-throughput LC-FLR-IMS-MS workflow in positive ion mode. We obtained IMS data from a selection of RapiFluor-MS (RFMS) labeled N-glycans and also glycopeptides. We describe how IMS is able to distinguish isomeric N-glycans and glycopeptides using both intact IMS and fragment-based IMS glycan sequencing experiments in positive ion mode, without significantly altering the high-throughput nature of the analysis. For the first time, we were able to successfully use IMS in positive ion mode to determine the branching of isomeric glycopeptides and RFMS labeled glycans. Further, we highlight that IMS glycan sequencing of fragments obtained from RFMS labeled glycans was similar to that of glycopeptides. Finally, we show that the IMS glycan sequencing approach can highlight shared structural features of nonisomeric glycans in a high-throughput LC-FLR-IMS-MS workflow.

Investigation of the effect of salt additives in Protein L affinity chromatography for the purification of tandem single-chain variable fragment bispecific antibodies.

Tandem single-chain variable fragment (scFv) bispecific antibodies (bsAb) are one of the most promising bsAb formats reported thus far. Yet, because of their increased aggregation propensity, high impurity content due to low expression level, smaller size and lack of the Fc region, it is challenging to isolate these products with high yield and purity within a limited number of purification steps in a scalable fashion. A robust purification process that is able to circumvent these issues is therefore of critical importance to allow effective isolation of this group of antibodies. We investigated the addition of sodium chloride (NaCl), calcium chloride (CaCl2), and L-arginine monohydrochloride (Arg·HCl) to the elution buffer of Protein L affinity chromatography, and propose here a novel mechanism for the modification of Protein L binding avidity that can lead to enhanced high molecular weight (HMW)-monomer separation, a preferential strengthening effect of the HMW-Protein L interaction compared to the monomer-Protein L interaction. In particular, we found Arg·HCl to be the most effective salt additive in terms of purity and recovery. The mechanism we propose is different from the widely reported chaotropic effect exerted by salt additives observed in Protein A chromatography. We also demonstrate here that a final eluate containing <1% HMW species and <100 ppm host cell proteins can be obtained within a two-step process with an overall yield of 65%, highlighting the promising suitability of Protein L affinity chromatography for the purification of kappa light chain-containing tandem scFv bsAb.

Application of maltose as energy source in protein-free CHO-K1 culture to improve the production of recombinant monoclonal antibody.

Oligosaccharides are generally considered to be un-utilized for growth of mammalian cells because their permeability across the cell membrane is low. However, in our previous study, we discovered that CHO and HEK293 cells consume maltose in culture media without serum and glucose. This is interesting because the transporter for maltose in mammalian cells has not been discovered to-date, and the only animal disaccharide transporter that is recently discovered is a sucrose transporter. The application of oligosaccharides in mammalian cell-based biopharmaceutical manufacturing can be beneficial, because it can theoretically increase carbohydrate content of the culture medium and decrease lactate production. Here, we first determined that specific maltose consumption rate in CHO cells was similar to galactose and fructose at 0.257 ng/cell/day. We then demonstrated that CHO cells can be cultivated with reasonable cell growth using higher maltose concentrations. After which, we evaluated the use of maltose supplementation in the production of a recombinant monoclonal antibody in batch and fed-batch cultures, demonstrating improvements in recombinant monoclonal antibody titer of 15% and 23% respectively. Finally, glycosylation profiles of the antibodies were analyzed.

[Effect of Qingyi Granule on HMGB1 Expression in Liver and Renal Tissues of Severe Acute Pancreatitis Rats].

OBJECTIVE: To explore the effect of Qingyi Granule (QYG) on high mobility group box-1 (HMGB1) expressions in liver and renal tissues of severe acute pancreatitis (SAP) rats. METHODS: Fifty-four Sprague-Dawley (SD) rats were divided into the sham-operation (SO) group, the SAP group, and the QYG group according to random digits table. Rats in the SAP group were induced by injecting 5% sodium taurocholate (STC). Liver and renal pathological changes were observed by HE staining. Serum contents of amylase (AMS), MDA, IL-1, and HMGB1 were detected by ELISA. HMGB1 protein expressions in liver and renal tissues were tested by immunohistochemistry. HMGB1 mRNA expressions in liver and renal tissues were detected by reversed transcription PCR. RESULTS: The pathological scores, serum levels of AMS, MDA, IL-1 and HMGB1, and protein and mRNA HMGB1 expressions in liver and renal tissues were increased more obviously in the SAP group than in the SO group (P < 0.05, P < 0.01). All of them could be down-regulated by QYG intervention, with the most significant effect seen at 72 h (P < 0.05, P < 0.01) in a time-effect relationship. CONCLUSIONS: HMGB1 participated in SAP complicated liver and renal injuries. QYG could effectively inhibit HMGB1 expressions, thereby attenuating SAP complicated liver and renal injuries.

[Differentially expressed proteins of severe acute pancreatitis intervened by Qingyi granule].

OBJECTIVE: To observe the effects of Qingyi Granule (QYG) on the changes of total protein expressions in the pancreatic tissue of rats with severe acute pancreatitis (SAP) induced by sodium taurocholate (STC). METHODS: SAP was induced by retrograded injecting 5% STC from the gut-pancreatic duct in 36 Sprague-Dawley (SD)rats. Then they were randomly divided into the SAP group and the QYG treatment group (abbreviated as the QYG group), 18 in each group. After successful modeling, rats in the QYG group were administered with QYG water solution (W: W = 1:1) once with an interval of 12 h (1 mL/100 g), while rats in the SAP group were administered with normal saline. The medication was performed four times. The total proteins were extracted from the pancreatic tissue of all rats to perform two-dimensional electrophoresis, fluorescent staining, and atlas analysis. The protein dots with differential expressions more than four times between each other in 48 h gel pictures were chosen and used for MALDI-TOF/TOF mass chromatographic analysis and biological information analysis. RESULTS: The 5% STC induced SAP model rats had typical pathological changes in the pancreatic tissue. The proteomics changes of the pancreatic tissue were analyzed by gel image manipulation software. Twenty two disparate points were detected between two groups at 48 h, 5 points of the protein were up-regulated and 17 points were down-regulated of the total after QYG intervention. Nine protein spots expressed differently more than 4 times and stably at 48 h, 7 kinds of proteins have been identified by mass chromatographic analysis and Data Base Retrieval, and they were Serpinb1a 39 kDa protein, Serpinb1a 43 kDa protein, Prdx4 Prx IV, Clps, gamma-actin (Actg1), Eprs and Hadhsc. Those proteins were involved in signal transmit during the process of SAP pancreas--pathological injury analyzed from their functions. CONCLUSIONS: Proteomics can well reflect the effects of QYG on differential expression proteins in the pancreatic tissue of rats with SAP. Studying differential expression proteins may provide a new theoretical basis and molecule target for QYG treating SAP.

Void exclusion of antibodies by grafted-ligand porous particle anion exchangers.

We describe a new variant of anion exchange chromatography in columns packed with porous particles that embody charged low-density polymer zones supported by a higher density polymer skeleton. IgG defies the norms of anion exchange and is excluded to the void volume at pH 3-10 and 0-4M NaCl. Void exclusion also occurs with Fab, F(ab')2, and IgM. Host cell protein contaminants mostly follow the usual norms of anion exchange and bind more strongly with increasing pH and decreasing conductivity. Sample buffer composition has no impact on partitioning so long as applied sample volume does not exceed the interparticle void volume of the column. Void-excluded antibody elutes in equilibration buffer. This seemingly conflicted collection of behaviors is reconciled by a variable size exclusion function mediated through the low-density polymer zones, the charge properties of the antibody species, and the pH and conductivity of the equilibration buffer. Current-generation porous particle anion exchangers that employ grafting techniques to achieve high charge density mediate void exclusion to varying degrees, with the best-suited achieving complete exclusion, and others as little as 65%. Perfusive and non-grafted particle-based exchangers mediate as little as 50% exclusion. Monoliths mediate no exclusion, due to their lack of an interparticle void volume. On qualified exchangers, the technique supports greater than 99% reduction of host proteins, DNA, and endotoxin. Virus is reduced more than 99.9%, and aggregates are reduced to less than 0.05%. The method supports better process control than other anion exchange formats because pH excursions in conjunction with changes in salt concentration do not occur until after the antibody has eluted from the column.

Principles and applications of steric exclusion chromatography.

We introduce a chromatography method for purification of large proteins and viruses that works by capturing them at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG). No direct chemical interaction between the surface and the target species is required. We refer to the technique as steric exclusion chromatography. Hydroxyl-substituted polymethacrylate monoliths provide a hydrophilic surface and support convective mass transport that is unaffected by the viscosity of the PEG. Elution is achieved by reducing PEG concentration. Selectivity correlates with molecular size, with larger species retained more strongly than smaller species. Retention increases with PEG size and concentration. Salts weaken retention in proportion to their concentration and Hofmeister ranking. Retention is enhanced near the isoelectric point of the target species. Virus binding capacity was measured at 9.9×10(12) plaque forming units per mL of monolith. 99.8% of host cell proteins and 93% of DNA were eliminated. Mass recovery exceeded 90%. IgM capacity was greater than 60 mg/mL. 95% of host cell proteins were eliminated from IgM produced in protein-free media, and mass recovery was up to 90%. Bioactivity was fully conserved by both viruses and antibodies. Process time ranged from less than 30 min to 2 h depending on the product concentration in the feed stream.

The association between problematic cellular phone use and risky behaviors and low self-esteem among Taiwanese adolescents.

BACKGROUND: Cellular phone use (CPU) is an important part of life for many adolescents. However, problematic CPU may complicate physiological and psychological problems. The aim of our study was to examine the associations between problematic CPU and a series of risky behaviors and low self-esteem in Taiwanese adolescents. METHODS: A total of 11,111 adolescent students in Southern Taiwan were randomly selected into this study. We used the Problematic Cellular Phone Use Questionnaire to identify the adolescents with problematic CPU. Meanwhile, a series of risky behaviors and self-esteem were evaluated. Multilevel logistic regression analyses were employed to examine the associations between problematic CPU and risky behaviors and low self-esteem regarding gender and age. RESULTS: The results indicated that positive associations were found between problematic CPU and aggression, insomnia, smoking cigarettes, suicidal tendencies, and low self-esteem in all groups with different sexes and ages. However, gender and age differences existed in the associations between problematic CPU and suspension from school, criminal records, tattooing, short nocturnal sleep duration, unprotected sex, illicit drugs use, drinking alcohol and chewing betel nuts. CONCLUSIONS: There were positive associations between problematic CPU and a series of risky behaviors and low self-esteem in Taiwanese adolescents. It is worthy for parents and mental health professionals to pay attention to adolescents' problematic CPU.

A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer?

Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.