Fragmentation Chain Research Articles

Abstract 337: Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature

Abstract Granzyme B is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-VEGF121) composed of granzyme B, VEGF121 (a naturally-occurring isoform of the VEGF-A family that binds to VEGF receptors R-1 and R-2), and a human IgG heavy chain fragment (for dimerization and to augment in vivo circulation and anti-tumor efficacy). GrB-Fc-VEGF121 was transiently expressed in HEK-293E cells under serum-free conditions purified with a final yield of approximately 40 mg/L. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-VEGF121 was comparable to that of commercially available human granzyme B. GrB-Fc-VEGF121 readily internalized into VEGFR-2+ cells within 2 hours of treatment while untargeted granzyme B was not internalized, demonstrating that internalization was receptor-mediated. Cytotoxicity studies against a panel of tumor and endothelial cells indicated cytotoxicity in the nanomolar range against cell lines expressing high levels of VEGFR-1 or VEGFR-2 while control (receptor-negative) cells demonstrated IC50 levels in the high micromolar range. Treatment of VEGFR-2+ endothelial and tumor cells with GrB-Fc-VEGF121 resulted in robust cell death via apoptosis and/or necrosis. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall loss of about 50% over 96 hours. The maximum tolerated dose of GrB-Fc-VEGF121 was not reached when BALB/c mice were treated with a total 475 mg/kg given every other day over 5 doses. Treatment of mice bearing OVCAR8 tumor xenografts with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. Treated tumors showed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells compared to controls, when assessed at the end of the study. These studies clearly indicate that GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. The tumor- and vascular-targeted agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 337.

Unusual High-Frequency Mechanical Properties of Polymer-Grafted Nanoparticle Melts.

Brillouin light spectroscopy is used to measure the elastic moduli of spherical polymer-grafted nanoparticle (GNP) melts as a function of chain length at fixed grafting density (0.47 chains/nm^{2}) and nanoparticle radius (8nm). While the moduli follow a rule of mixtures (Wood's law) for long chains, they display enhanced elasticity and anomalous dissipation for graft chains <100 kDa. GNP melts with long polymers at high σ have a dry zone near the GNP core, surrounded by a region where the grafts can interpenetrate with chain fragments from adjacent GNPs. We propose that the departures from Wood's law for short chains are due to the effectively larger silica volume fraction in the region where sound propagates-this is caused by the short, interpenetrated chain fragments being pushed out of the way. We thus conclude that transport mechanisms (of gas, ions, sound, thermal phonons) in GNP melts are radically different if interpenetrated chain segments can be "pushed out of the way" or not. This provides a facile new means for manipulating the properties of these materials.

Ultrastructural behavior of cell wall polysaccharides

Studies on the behavior of wood polysaccharides inside and outside the cell wall were carried out under varying conditions using the electron microscope in combination with physico-chemical methods to gain new information on the super- molecular structure of the polysaccharides. Several fractions containing different polyoses were obtained by fractionation of the alkali extract of holocellulose. Isolated polyoses are at least partially able to arrange themselves in fibrillar elements. After delignification, the cellulose is recognizable within the cell wall as microfibrils with an average diameter of about 250 À. However, these fibrillar units seem to be rather unstable as they can easily be split into units 120 À in diameter by chemical treatment. After partial hydrolysis, isolated cellulose shows more subunits about 30 A in diameter at higher magnification. Moreover, the more sensitive portions of the fibrils in the longitudinal direction are attacked by partial hydrolysis, and these portions seem to grow with increasing duration of the hydrolysis. Fractionation of cellulose isolated from thermally treated wood shows that the chain fragments are multiples about 300 À in length, A tentative model of the supermolecular arrangement of the cellulose in relation to the other cell wall components is presented.

Brain Penetrant Humanized scFv Antibody Block of P2 × 4 Receptor for Treatment of Chronic Pain

Our collaborative efforts are focused on validation of promising humanized single chain Fragment variable (hscFv) antibodies targeting a unique amino acid peptide sequence of P2 × 4 receptor (P2 × 4R). Data indicate single dose post-treatment with our newly engineered hP2 × 4 scFv antibody restores mechanical and cold pain-related behaviors to baseline in our trigeminal nerve compression model (FRICT-ION). The reversal to naïve baseline persists until experiment end at 10 weeks. Anxiety-like behaviors that develop in mice with chronic pain do not develop in hscFv antibody treated mice. We are characterizing the efficacy of lead P2 × 4R hscFv antibodies on neuronal cell-type specific activation patterns in medullary spinal V nucleus slices from Fos-TRAP(targeted recombination in active populations) mice and cultured trigeminal ganglia (TG) neurons from FRICT-ION mice. Engineered scFv antibodies feature binding affinity and activity similar to or better than monoclonal antibodies because they are much smaller in size. Thus, they are brain/nervous tissue penetrant due to their small size and have promising biotherapeutic applications for both the nervous and immune systems, now recognized as interactive in chronic pain. The ribosome display method utilized to generate the scFvs is a powerful cell-free technology widely used to select single-chain antibody fragments against the target of choice. This method is inexpensive, rapid, and was used to quickly develop repertoires of high-affinity antibodies targeting P2 × 4. In our hands the scFvs developed have high affinity in the picomolar range, superior stability and solubility. The patent was filed and published. Moving forward we currently continue evaluating these humanized P2 × 4R scFv antibodies that have strong nanomolar binding affinity, reduced self-immunogenicity, and relatively inexpensive large-scale production toward new drug application status based on the successful reversal of pain related measures and reduction of neuronal activation. This project opens new avenues for development of highly effective non-opioid biologic therapeutic interventions for chronic pain. Grant support from DoD grant W81XWH2010930 (KNW, SRAA); VA Merit grant BX002695 (KNW); NIH NIDCR R21 DE028096; NIH UG3 NS123958.

Virus‐induced Hepatic Expression of an Oxidized Phospholipid‐binding Antibody Fragment Prevents Initiation of Hepatic Steatosis and Progression to Fibrosis

Oxidized phospholipids have been implicated in chronic pathologies. It had been shown that hyperlipidemic LDL‐R‐deficient mice transgenic for a single chain fragment of an oxidized phosphatidylcholine (OxPC)‐recognizing IgM E06 (scFv‐E06) were protected from developing non‐alcoholic fatty liver disease (NAFLD). To investigate the role of OxPCs in initiation of diet‐induced hepatic steatosis as well as independently in progression to fibrosis, we used an adeno‐associated virus serotype 8 to induce hepatic expression scFv‐E06 in a cre‐dependent manner (AAV8‐scFv‐E06) in B6. Speer6‐ps1Tg(Alb‐cre)21Mgn/J(Alb‐cre) mice. Virus‐induced expression of scFv‐E06 protected mice from diet‐induced hepatic steatosis by reducing liver triglyceride content and tissue damage as measured by plasma liver enzymes. Separately, expression of scFv‐E06 in mice with established hepatic steatosis prevented the progression to hepatic fibrosis. We developed a targeted, mass spectrometry‐based oxophospholipidomic platform to identify and measure OxPC species that were potentially altered by expression of scFv‐E06. We discovered that several OxPCs in the plasma were significantly reduced by scFv‐E06 expression in both steatosis and fibrosis and identified unique OxPC species that were present in both disease settings. Additionally, we show that the identified OxPC species reduced by scFv‐E06 expression dysregulate gene expression and metabolic function of hepatocytes and hepatic stellate cells in vitro. Together, we show that virus‐induced hepatic expression of scFv‐E06 is sufficient to prevent hepatic steatosis as well as intervene in progression from hepatic steatosis to hepatic fibrosis. Furthermore, we identified unique plasma OxPC species that are reduced by expression of scFv‐E06 and may drive initiation of hepatic steatosis and independently progression to hepatic fibrosis. These data demonstrate an important role for individual OxPCs in NAFLD and may provide clinically relevant biomarkers for diagnosis of hepatic steatosis and fibrosis.

Development of SARS-CoV-2 specific neutralizing recombinant monoclonal antibodies

Abstract Development of effective therapeutic strategies to combat COVID-19 relies on understanding the cellular and molecular mechanisms of infection. Antibodies specific to SARS-CoV-2 aid in accelerating research to characterize viral protein function and immune response to infection. An urgent need for SARS-CoV-2 specific antibodies arose, as several antibodies already available in the market cross-reacted with other members of the coronavirus family. To address this need for specificity and urgency, we collaborated with a leading pharmaceutical company to develop antibodies specific to SARS-CoV-2. We generated a single chain fragment variable (scFv) library for SARS-CoV-2 using computational expansion of published antibody structures against SARS-CoV-1. This enabled us to make recombinant antibodies in record time of 3 months. These synthetic sequences were grafted onto both human and human-rabbit chimeric Fc enabling use of these antibodies in a multitude of immunoassays. Our antibodies were targeted against the SARS-CoV-2 spike protein; the receptor binding domain in the spike protein plays a vital role in viral entry into host cells and subsequent infection, making it an important target for neutralizing antibodies and antiviral treatments. Some of our antibodies demonstrate neutralizing capabilities, making them a valuable tool to study neutralizing antibody response to infection. These antibodies are specific to SARS-CoV-2 and do not cross-react with other coronaviruses. The specificity and versatility of our antibodies enables scientists to better understand SARS-CoV-2 and potentially aid in developing therapeutic strategies against COVID-19 which continues to be important as newer variants of the virus emerge.

Discovery and structure-activity relationship studies of novel Bcl-2/Mcl-1 dual inhibitors with indole scaffold

The Bcl-2 anti-apoptotic proteins were widely overexpressed in diverse tumor cells, especially for Bcl-2 and Mcl-1, which regarding as important targets of apoptosis induction and resistance of chemotherapy. We identified novel Bcl-2/Mcl-1 dual inhibitors with indole scaffold by the optimization of hit 1. Structure modification against several moieties including hydrophobic fragment, side chain and benzoic acid fragment was conducted and the structure-activity relationship was analyzed. The representative compound 12f exhibited sub-micromolar binding affinities to Bcl-2/Mcl-1 without binding affinity to Bcl-XL. Mechanism of action studies suggested that compound 12f dose-dependently triggered apoptosis in HL-60 cells. Compound 12f represents a novel Bcl-2/Mcl-1 dual inhibitor which deserving further study.

Immunoglobulin light chain amyloidosis: 2022 update on diagnosis, prognosis, and treatment.

Immunoglobulin light chain amyloidosis is a clonal, nonproliferative plasma cell disorder in which fragments of immunoglobulin light or heavy chain are deposited in tissues. Clinical features depend on organs involved but can include heart failure with preserved ejection fraction, nephrotic syndrome, hepatic dysfunction, peripheral/autonomic neuropathy, and "atypical smoldering multiple myeloma or monoclonal gammopathy of undetermined significance (MGUS)." Tissue biopsy stained with Congo red demonstrating amyloid deposits with apple-green birefringence is required for the diagnosis of AL amyloidosis. Invasive organ biopsy is not required in 85% of patients. Verification that amyloid is composed of immunoglobulin light chains is mandatory. The gold standard is laser capture mass spectroscopy. N-terminal pro-brain natriuretic peptide (NT-proBNP or BNP), serum troponin T (or I), and difference between involved and uninvolved immunoglobulin free light chain values are used to classify patients into four groups of similar size; median survivals are 73, 35, 15, and 5months. All patients with a systemic amyloid syndrome require therapy to prevent deposition of amyloid in other organs and prevent progressive organ failure. Current first-line therapy with the best outcome is daratumumab, bortezomib, cyclophosphamide, and dexamethasone. The goal of therapy is a complete response (CR). In patients failing to achieve this depth of response options for consolidation include pomalidomide, stem cell transplantation, venetoclax, and bendamustine. Delayed diagnosis remains a major obstacle to initiating effective therapy prior to the development of end-stage organ failure. Trials of antibodies to catabolize deposited fibrils are underway.

Engineering a single-chain variable fragment of cetuximab for CAR T-cell therapy against head and neck squamous cell carcinomas

The monoclonal antibody cetuximab recognizes domain III of the epithelial growth factor receptor (EGFR) with high-affinity and is an important element in the treatment of several malignancies that overexpress non-mutated wild-type EGFR. In order to create an EGFR recognizing chimeric antigen receptor (CAR) for cellular immunotherapy of head and neck squamous cell carcinoma (HNSCC), we rationally designed single chain fragments of different lengths based on the cetuximab variable heavy and light chains. We then cloned the different cetuximab fragments into our second generation CAR construct, expressed CARs on primary human T-cells from healthy donors using mono- or biscistronic lentiviral vectors and tested the stability, functionality and specificity of the CARs. Our smallest CAR construct was most efficient with greatly improved vector production and T-cell transduction efficacy. Finally, we demonstrated that the new cetuximab CAR construct expressed on T-cells is highly reactive against EGFR-positive HNSCCs and also malignant cells from other solid cancer entities. In conclusion, we generated an optimized high-affinity EGFR CAR construct for the next steps in cancer immunotherapy, which need to focus on the development of armored CAR T-cells that will be more resistant and effective in the hostile microenvironment present in solid cancers.

GENERALIZED MATHEMATICAL MODEL OF MONEY LAUNDERING AND TERRORISM FINANCING RISK TYPOLOGY IN BIG DATA OF SOCIO-ECONOMIC SYSTEMS

The big data of socio-economic systems is investigated in order to synthesize a generalized model of the typology of the risk of money laundering. The analysis of actual requirements to the typology model was carried out. A model for input, output and classification of objects, sets, categories in big data represented by topos has been developed. It is shown that classified objects can be entered into an artificial intelligence database and used to search and analyze typologies of money laundering risks. The concept is introduced and the category-theoretic formula of the event of the risk of money laundering is synthesized. In addition, a retrospective event was singled out and its theoretical justification was carried out. A method for fragmentation of chains of Markov occurrences is proposed and analyzed. Within the framework of the category-theoretic approach, chains are represented by category compositions. A new scheme developed instead of the well-known scheme of the normal Markov algorithm is analyzed. In the scheme, locally final occurrences were discovered and theoretically substantiated, which were not taken into account and were not used by Markov. Locally final occurrences in the scheme of the Markov algorithm are found in parallel chains of occurrences and voids. It has been established that locally final substitutions play an important role in the search and analysis of typologies of money laundering risks in big data of socio-economic systems. It has been determined that typologies of money laundering risks can be represented as categories of sets and n-categories. A formula for an incomplete risk typology has been developed. It is concluded that the synthesized model of the typology of money laundering risks provides for the detection and analysis of local typologies of individual risks.

Modified concept of bond blocks and its applications

The essence of the method for kinetic modeling and CFD calculations of polymer synthesis is presented. The method is named as the modified concept of bond blocks, because species conservation equation are written for polymer chain fragments – bond blocks. For the first time it was shown that it is necessary for all elements of the polymer chains to be represented by elementary bond block and one elementary bond block should not include other elementary bond blocks. For the first time an algorithm has been demonstrated for calculating the molar mass distribution of a polymer based on information of the concentrations of elementary bond blocks (for instance, poly(methyl methacrylate)). The modified concept of bond blocks allows performing CFD calculations by working directly with the CFD simulators interface without writing additional program code. The modified concept of bond blocks is of interest to readers, as it simplifies the technical side of CFD calculations of polymer synthesis. The modified concept of bond blocks was used for the first time to calculate the cationic copolymerization of isobutylene with isoprene in tubular turbulent diffuser-confuser reactors with various feed type of monomers and catalyst solution. It was shown that the highest conversion of monomers can be achieved when fractional feed of catalyst and single-point feed of monomers. For the first time, a method has been proposed for accurate calculation of the shrinkage of a composite material using a modified concept of bond blocks, given the content of associates in a thermosetting binder. This method is useful for controlling the dimensional accuracy of composite products and the designing of technological equipment in which these products are molded.

Preparation of thermoplastic starch with comprehensive performance plasticized by citric acid

AbstractTo prepared thermoplastic starch (TPS) with good thermal plasticization processibility and resistance to retrogradation is important in this field. Herein, TPS plasticized with different plasticizers were successfully produced using a twin‐screw‐extruder. Effects of glycerol, xylitol, and citric acid on the retrogradation properties of TPS were studied by differential scanning calorimetry (DSC) and X‐ray diffraction (XRD). It showed that citric acid can improve the thermal plasticization processibility of TPS through three mechanisms, that is, chemical reaction between citric acid and starch, chain fragmentation of starch molecule due to the acidolysis of citric acid, and decrease of interaction between starch molecular chains. Since citric acid can be chemically fixed in the starch, excellent retrogradation resistance was achieved. After the accelerated retrogradation at 60°C/80RH for 48 h, the retrogradation and crystallinity of CA4TPS are 0.8% and 4.26%, respectively, which were lower than TPS without citric acid. In this work, a series of TPS samples with citric acid content from 0 to 8 wt% were prepared, and the DSC and XRD results showed that the TPS with 4 wt% citric acid yielded the least amount of retrogradation. The results of this investigation lay a solid foundation for the preparation and application of TPS with excellent performance.

A convergent total synthesis of resorcylic acid lactones zeaenol and cochliomycin A

A convergent approach for the stereoselective total synthesis of zeaenol and cochliomycin A is reported. The key steps involved are alkyne generation using Ohira-Bestmann reagent onto sugar lactol, NaH mediated one-pot esterification and acetonide deprotection, Trost’s protocol of hydrosilylation followed by protodesilylation to get exclusively E-olefin from internal alkyne and ring closing metathesis (RCM) reaction. Readily accessible d-lyxose has been utilized as the chiron substrate for generation of aliphatic chain fragment and 2,4,6-trihydroxybenzoic acid has been used for the synthesis of vinylated arene fragment.

Фрагментация и консолидация производственных цепочек в мировой редкоземельной промышленности

The article analyzes the issues of fragmentation and consolidation of value chains using the example of the global rare earth industry. On the one hand, there is a tendency in the world towards specialization of companies at technological stages, which is dictated by the laws of market economy. On the other hand, such fragmentation in the rare earth industry made it possible to monopolize the market and concentrate most of the production stages within one country. Governments of the Western countries are extremely concerned about this problem, but so far only options are being proposed for its solution, which are based on the cooperation of individual participants in the technological chain. The paper discusses consolidation of efforts on the global scale aimed at reducing China's influence on the rare earth metals market, often in spite of the market efficiency. Russia has a significant raw material potential and processing technologies created during the Soviet period, but is not yet able to transform the raw material potential into a high-tech one due to insufficient domestic demand for highly purified rare earth metals

Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product.

Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5–10% hospitalization and 2–3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin-converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N-linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1) size heterogeneity analysis to determine the presence/absence of the non-glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2) capillary gel isoelectric focusing for possible N-glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3) capillary gel electrophoresis for N-glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N-glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N-linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule.

Haemagglutinin antigen selectively targeted to chicken CD83 overcomes interference from maternally derived antibodies in chickens

Maternally derived antibodies (MDAs) are important for protecting chickens against pathogens in the neonatal stage however, they often interfere with vaccine performance. Here, we investigated the effects of MDAs on a targeted antigen delivery vaccine (TADV), which is developed by conjugating H9 subtype avian influenza virus haemagglutinin (HA) antigen to single chain fragment variable (scFv) antibodies specific for the chicken antigen presenting cell receptor CD83. Groups of 1-day-old chickens carrying high levels of MDAs (MDA++) and 14-day old chickens carrying medium levels of MDAs (MDA+) were immunised with TADV (rH9HA-CD83 scFv), untargeted rH9HA or inactivated H9N2 vaccines. Immunogenicity in these vaccinated chickens was compared using haemagglutination inhibition (HI) and enzyme-linked immunosorbent assays (ELISA). The results showed that the TADV (rH9HA-CD83 scFv) induced significantly higher levels of H9HA-specific antibody titres compared to the untargeted rH9HA and inactivated H9N2 vaccines in MDA++ and MDA+ chickens. Overall, the data demonstrates immune responses induced by TADV are not affected by the MDA in chickens.

BROLUCIZUMAB - A NEW PLAYER IN THE FIELD OF ANTI-VEGF THERAPY OF NEOVASCULAR AGE-RELATED MACULAR DEGENERATION. A REVIEW.

Vascular endothelial growth factor (VEGF) has been identified as a major promoter of the development of choroidal neovascularization in age-related macular degeneration. The development of choroidal neovascularization can be slowed by preventing the binding of vascular endothelial growth factor to cellular VEGF receptor-2 present on vascular endothelial cells, which represents the major proangiogenic stimulus. Advances in the development of anti-VEGF therapy have led to significant improvement in visual acuity outcomes in recent years that neovascular age-related macular degeneration can no longer be considered an incurable disease. Despite its many advantages, the current standard of care, which is the frequent application of VEGF blockers to the vitreous, is a significant burden on both the patient and the healthcare system. This review is aim on a new brolucizumab molecule (also known as RTH 258 or formerly ESBA 1008). The article focuses on the molecular aspects of the drug and an overview of the basic preclinical and clinical studies that were performed during drug development. Brolucizumab is a single chain fragment of a humanized monoclonal antibody with a molecular weight of 26 kDa that inhibits VEGF-A. Preclinical animal studies have shown good penetration of the molecule through the retina with minimal systemic exposure. The SEE study (phase 1/2) demonstrated safety and tolerability after drug administration. The OSPREY (phase 2) study demonstrated the same efficacy of brolucizumab on visual acuity in the 8-week dosing regimen compared to aflibercept. In the same study, patients were also pilot tested in a 12-week dosing regimen. The HAWK and HARRIER studies (phase 3) demonstrated the efficacy of the drug at a dose of 6 mg in a 12-week dosing schedule in 55.6 % and 51 % of patients, respectively.

Online Electrochemical Reduction of Both Inter- and Intramolecular Disulfide Bridges in Immunoglobulins.

Electrochemical reduction of intermolecular disulfide bridges has previously been demonstrated in immunoglobulins but failed to achieve reduction of intramolecular bonds. We now report an improved method that achieves the full reduction of both intermolecular and intramolecular disulfide bridges in a set of monoclonal antibodies based on their intact mass and on MS/MS analysis. The system uses an online electrochemical flow cell positioned online between a chromatography system and a mass spectrometer to give direct information on pairs of heavy and light chains in an antibody. The complete reduction of the intramolecular disulfide bridges is important, as the redox state affects the intact mass of the antibody chain. Disulfide bonds also hamper MS/MS fragmentation of protein chains and thus limit the confirmation of the amino acid sequence of the protein of interest. The improved electrochemical system and associated protocols can simplify sample processing prior to analysis, as chemical reduction is not required. Also, it opens up new possibilities in the top-down mass spectrometry analysis of samples containing complex biomolecules with inter- and intramolecular disulfide bridges.

Analysis of glycan ratio of Chinese hamster ovary cell-cetuximab antigen-binding segment via rapid enzyme digestion with endo-β-N-acetylglucosaminidase F

西妥昔单抗具有较复杂的糖基化修饰,在抗原结合片段(Fab)和可结晶片段(Fc)的重链上都含有2个N-糖基化位点,其中Fab段的糖基化最为复杂,要研究清楚该位点的糖基化修饰,开发专一性切糖技术和稳定的聚糖比例分析方法是当前迫切需要解决的难题。以中国仓鼠卵巢(CHO)细胞表达的西妥昔单抗为研究对象,使用β-N-乙酰氨基葡萄糖苷酶(Endo F2)开发了一种快速Fab段聚糖释放的方法,利用超高效液相色谱-高分辨质谱(UPLC-HRMS)进行了定性和聚糖比例分析。第一步对抗体原液进行非变性酶切,抗体原液经超纯水稀释后,加入糖苷酶Endo F2进行酶切,通过质谱对质量数的解析,结果表明Endo F2酶切时间5 min, Fab段的聚糖就能完全切除,而Fc段的聚糖不受影响,实现了快速酶切,而且切糖具有很好的专一性。第二步对Fab段聚糖进行比例分析,将释放的聚糖经对氨基苯甲酰胺(2-AB)荧光标记后使用超高效液相色谱联用荧光检测器(FLR)进行检测,在亲水作用色谱(HILIC)柱上得到良好的分离并可以进行稳定地聚糖比例分析。3次独立试验结果表明,酶切后的质谱图基本一致,且聚糖的比例结果也基本一致,表明Endo F2酶切方法和聚糖比例分析方法都具有较好的稳定性和可靠性。此外,通过测定来自两个不同工艺生产的样品,数据显示两者的糖谱上具有非常明显的差异,表明利用开发的方法可以实现对抗体生产工艺进行监测研究,对抗体生产工艺的评估具有非常重要的意义。

Spectroscopic analysis of metal ions effect on the aggregation of a λ6aJL2R24G protein variant

Light-chain amyloidosis (AL) is one of the most common forms of amyloidosis. It is characterized by the extracellular deposition of variable domain fragments of immunoglobulin light chains. The clinical manifestations of this disease vary because the deposits can be found in almost all the organs of the human body causing various failures. Approximately 30% of AL cases contain a λ6a germline and 25% of those cases presents a Glycine at position 24 instead of Arginine (R24G). This mutation causes the protein to be thermodynamically more unstable and to be more amyloidogenic.