Enhanced efficacy of glycoengineered rice cell-produced trastuzumab.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 µg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

Flexible Piezoelectric and Pyroelectric Nanogenerators Based on PAN/TMAB Nanocomposite Fiber Mats for Self-Power Multifunctional Sensors.

Self-powered wearable electronics to convert mechanical and thermal energy into electrical energy are important for biomedical monitoring, which highly require good flexibility, comfortability, signal sensitivity, and accuracy. In this work, composite nanofiber mats of polyacrylonitrile (PAN) and trimethylamine borane (TMAB) were prepared by electrospinning, which exhibited excellent piezoelectric and pyroelectric abilities in harvesting mechanical and thermal energy. The PAN/TMAB-4 nanofiber mats not only generated a high voltage of up to 2.56 V and a high power of 0.19 µW upon shape deformation but also exhibited linear voltage response to thermal gradient. The hybrid piezoelectric and pyroelectric output signals were successfully integrated together and have been applied to precisely monitor human vital signs, including elbow bending angles, foot posture, and breathing status, in real time by attaching the flexible sensors to proper human body parts. Overall, good flexibility, bifunctional sensing ability, and self-power make PAN-/TMAB-type sensors very attractive in fabricating high-performance electronics for detecting motion, monitoring health, and making portable microelectronics.

Ocrelizumab quantitation by liquid chromatography-tandem mass spectrometry.

Introduction: Ocrelizumab is a monoclonal anti-CD20 antibody approved for the treatment of multiple sclerosis (MS). The clinical value of therapeutic drug monitoring (TDM) for this antibody in treatment of MS is unknown, and an adequately specific and precise quantitation method for ocrelizumab in patient serum could facilitate investigation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitation methods have been shown to have higher analytic specificity and precision than enzyme-linked immunosorbent assays. Objectives: To establish and validate an LC-MS/MS-based quantitation method for ocrelizumab. Methods: We present an LC-MS/MS-based quantitation method using immunocapture purification followed by trypsinization and analysis by a triple quadrupole mass analyzer obtaining results within the same day. Results: We found that the ocrelizumab peptide GLEWVGAIYPGNGDTSYNQK (Q1/Q3 Quantifier ion: 723.683+/590.77 y112+ Qualifier ion: 723.683+/672.30 y122+) can be used for quantitation and thereby developed a method for quantifying ocrelizumab in human serum with a quantitation range of 1.56 to 200 µg/mL. The method was validated in accordance with EMA requirements in terms of selectivity, carry-over, lower limit of quantitation, calibration curve, accuracy, precision and matrix effect. Ocrelizumab serum concentrations were measured in three MS patients treated with ocrelizumab, immediately before and after ocrelizumab infusion, with additional sampling after 2, 4, 8 and 12 weeks. Measured serum concentrations of ocrelizumab showed expected values for both Cmax and drug half-life over the sampled time period. Conclusion: We have established a reliable quantitation method for serum ocrelizumab that can be applied in clinical studies, facilitating the evaluation of ocrelizumab TDM in MS.

Ravulizumab: Characterization and quantitation of a new C5 inhibitor using isotype specific affinity purification and high-resolution mass spectrometry.

INTRODUCTION: Ravulizumab (RAVUL) is a new complement inhibitor, with a difference of 4 amino acids in the heavy chain from a predecessor compound, eculizumab (ECUL). OBJECTIVES: First, to utilize mass spectrometry (MS) to characterize RAVUL and verify differences from its predecessor and, second, to validate and implement a lab developed test (LDT) for RAVUL that will allow for quantitative therapeutic monitoring. METHODS: A time-of-flight mass spectrometer (TOF-MS) was used to characterize and differentiate the molecular weight differences between RAVUL and ECUL by both digest and reduction experiments. In parallel, an LDT for RAVUL was validated and implemented utilizing IgG4 enrichment with light chain detection and quantitation on a high throughput orbitrap MS platform. RESULTS: The TOF-MS platform allowed for the mass difference between RAVUL and ECUL to be verified along with providing a proof of concept for a new intact protein quantitation software. An LDT on an orbitrap MS was validated and implemented using intact light chain quantitation, with the limitation that it cannot differentiate between ECUL and RAVUL. The LDT has an analytical measuring range from 5 to 600 mcg/mL, inter-assay imprecision of ≤13% CV (n = 13) and accuracy with <4% error from expected values (n = 20). CONCLUSION: The TOF-MS is a versatile development platform that can be used to characterize and verify the molecular weight differences between the ECUL and RAVUL heavy chains. Routine laboratory testing for RAVUL was viable using an orbitrap-MS to quantitate using the mass of the intact light chain. These two platforms, combined, provide incomparable value in development of LDTs for the clinical laboratory.

Preclinical pharmacokinetics of a recombinant humanized rabbit anti-VEGF monoclonal antibody in rabbits and monkeys.

TMAB001 is a humanized rabbit monoclonal antibody (mAb) designed to bind and neutralize human vascular endothelial growth factor (VEGF)-165. The purpose of the study was to investigate the pharmacokinetics (PK) and ocular tissue distribution after a single intravitreal (IVT) dose in rabbits and monkeys. Rabbits (2.5 mg/eye; n = 40) and monkeys (2.5 mg/eye; n = 12) received TMAB001 as a bilateral IVT dose. TMAB001 concentrations were measured in ocular tissues in all rabbits and monkeys by enzyme-linked immunosorbent assay (ELISA). TMAB001 and VEGF concentrations were measured in serum of monkeys by ELISA. Following a single bilateral IVT injection of TMAB001 2.5 mg/eye, the highest concentration was in vitreous humor, followed by retina and choroid, and the lowest concentration was in lens. In rabbits, TMAB001 was still detectable in ocular tissues at day 21 after single IVT dose, with the highest level in the vitreous humor and then retina, with longest t1/2 in aqueous humor and shortest t1/2 in choroid. In monkeys, tmax in serum was 43 h and t1/2 was approximately 5.5 days. Cmax in serum was much lower than that in vitreous, nearly 1/200. After IVT injection of TMAB001, total VEGF concentrations in serum and ocular tissues increased over time. VEGF concentration in retina and choroid increased over time, up to 336 h after administration. This study demonstrated that TMAB001 could reach the drug target sites-retina and choroid after a single bilateral IVT administration in rabbits and monkeys, with a long t1/2 in vitreous humor. TMAB001 also showed strong capability to neutralize VEGF. The study further confirmed that full-length antibodies can also efficiently diffuse and distribute in ocular tissues.

Assessment of thyroid function in children, adults and pregnant and lactating women after long-term salt iodisation measurements.

Universal salt iodisation (USI) has been successfully implemented in China for more than 15 years. Recent evidence suggests that the definition of 'adequate iodine' (100-199 µg/l) be revised to 'sufficient iodine' (100-299 µg/l) based on the median urinary iodine concentration (MUI) in school-age children. The objective of this study was to determine the prevalence of thyroid dysfunction in populations after long-term salt iodisation and examine whether the definition of adequate iodine can be broadened to sufficient iodine based on the thyroid function in four population groups. A cross-sectional survey was conducted in six provinces in the northern, central and southern regions of China. Four population groups consisting of 657 children, 755 adults, 347 pregnant women and 348 lactating women were recruited. Three spot urinary samples were collected over a 10-d period and blood samples were collected on the 1st day. In the study, among the adults, pregnant women and lactating women, the prevalence rates of elevated thyroglobulin antibody and thyroid microsomal antibody levels were 12·4, 8·5 and 7·8 %, and 12·1, 9·1 and 9·1 %, respectively. Abnormally high thyroid dysfunction prevalence was not observed after more than 15 years of USI in China because the thyroid dysfunction rates were all <5 %. The recommended range should be cautiously broadened from adequate iodine to sufficient iodine according to the MUI of school-age children considering the high levels of hormones and antibodies in the other populations. Adults, particularly pregnant women positive for thyroid antibodies, should be closely monitored.

Quantitative Peptidomics: General Considerations.

Peptidomics is the detection and identification of the peptides present in a sample, while quantitative peptidomics provides additional information about the amounts of these peptides. Comparison of peptide levels among two or more samples is termed relative quantitation. It is also possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards, which requires a separate standard for each peptide. In contrast, relative quantitation can compare levels of all peptides that are detectable in a sample, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available.

Quantitative Peptidomics with Isotopic and Isobaric Tags.

In differential peptidomics, peptide profiles are compared between biological samples and the resulting expression levels are correlated to a phenotype of interest. This, in turn, allows us insight into how peptides may affect the phenotype of interest. In quantitative differential peptidomics, both label-based and label-free techniques are often employed. Label-based techniques have several advantages over label-free methods, primarily that labels allow for various samples to be pooled prior to liquid chromatography-mass spectrometry (LC-MS) analysis, reducing between-run variation. Here, we detail a method for performing quantitative peptidomics using stable amine-binding isotopic and isobaric tags.

Fast and Reliable Quantitative Peptidomics with labelpepmatch.

The use of stable isotope tags in quantitative peptidomics offers many advantages, but the laborious identification of matching sets of labeled peptide peaks is still a major bottleneck. Here we present labelpepmatch, an R-package for fast and straightforward analysis of LC-MS spectra of labeled peptides. This open-source tool offers fast and accurate identification of peak pairs alongside an appropriate framework for statistical inference on quantitative peptidomics data, based on techniques from other -omics disciplines. A relevant case study on the desert locust Schistocerca gregaria proves our pipeline to be a reliable tool for quick but thorough explorative analyses.

Oxidative cleavage of DNA by pentamethine carbocyanine dyes irradiated with long-wavelength visible light.

Here we report the synthesis of seven symmetrical carbocyanine dyes in which two nitrogen-substituted benz[e]indolium rings are joined by a pentamethine bridge that is meso-substituted with chlorine or bromine versus hydrogen. The heteroatom of benz[e]indolium is modified with a phenylpropyl, methyl, or cationic quaternary ammonium group. In reactions containing micro molar concentrations of halogenated dye, irradiation at 575, 588, 623, or 700nm produces good photocleavage of plasmid DNA. UV-visible spectra show that the carbocyanines are in their H-aggregated and monomeric forms. Scavenger experiments point to the involvement of singlet oxygen and hydroxyl radicals in DNA photocleavage.

AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity.

Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the thimet oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides. Indeed, we have identified the peptide AGHLDDLPGALSAL (AGH), a fragment of the hemoglobin alpha-chain, which specifically bind to the inactive thimet oligopeptidase in the substrate capture assay. Previous peptidomics studies have identified the AGH as well as many other natural peptides derived from hemoglobin alpha-chain containing this sequence, further suggesting that AGH is a natural endogenous peptide. Pharmacological assays suggest that AGH inhibits peripheral inflammatory hyperalgesic responses through indirect activation of mu opioid receptors, without having a central nervous system activity. Therefore, we have successfully used the substrate capture assay to identify a new endogenous bioactive peptide derived from hemoglobin alpha-chain.

Analysis of peptides secreted from cultured mouse brain tissue.

Peptides represent a major class of cell-cell signaling molecules. Most peptidomic studies have focused on peptides present in brain or other tissues. For a peptide to function in intercellular signaling, it must be secreted. The present study was undertaken to identify the major peptides secreted from mouse brain slices that were cultured in oxygenated buffer for 3-4h. Approximately 75% of the peptides identified in extracts of cultured slices matched the previously reported peptide content of heat-inactivated mouse brain tissue, whereas only 2% matched the peptide content of unheated brain tissue; the latter showed a large number of postmortem changes. As found with extracts of heat-inactivated mouse brain, the extracts of cultured brain slices represented secretory pathway peptides as well as peptides derived from intracellular proteins such as those present in the cytosol and mitochondria. A subset of the peptides detected in the extracts of the cultured slices was detected in the culture media. The vast majority of secreted peptides arose from intracellular proteins and not secretory pathway proteins. The peptide RVD-hemopressin, a CB1 cannabinoid receptor agonist, was detected in culture media, which is consistent with a role for RVD-hemopressin as a non-classical neuropeptide. Taken together with previous studies, the present results show that short-term culture of mouse brain slices is an appropriate system to study peptide secretion, especially the non-conventional pathway(s) by which peptides produced from intracellular proteins are secreted. This article is part of a Special Issue entitled: An Updated Secretome.