Subunit mass analysis for monitoring antibody oxidation

Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.     

A Quadrupole Dalton-based multi-attribute method for product characterization,process development,and quality control of therapeutic proteins

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.     

Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles

Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N‐glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N‐glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under‐appreciated for these under‐utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511–522, 2017     

新疆典型盐生植物营养器官盐分积累与生态化学计量特征

对盐生植物体内器官盐分积累和碳(C)、氮(N)、磷(P)化学计量特征的研究, 有助于了解盐生植物养分、盐分的分配机制和其对盐渍环境的适应策略。该文选择新疆4种典型的真盐生植物(3种灌木植物: 盐节木(Halocnemum strobilaceum)、小叶碱蓬(Suaeda microphylla)、盐爪爪(Kalidium foliatum), 1种草本植物: 盐角草(Salicornia europaea)为研究对象, 对比研究了它们的根、茎、叶中的盐分积累和C、N、P化学计量特征以及二者间的相关性。结果显示: 1)在生长旺盛期, 4种盐生植物体内盐分积累可形成“盐岛”效应(Na⁺、Cl^-和电导率随盐离子从植物根部向顶端运输过程呈显著增加的趋势)。2) 4种盐生植物中灌木群落的生长主要受到P的限制; 草本群落的生长受N和P (偏P)共同限制。3)器官、物种和二者的交互作用均能影响盐生植物的盐分(除Mg²⁺外)特征和C、N、P生态化学计量特征。4) 4种盐生植物C与N、P之间显著负相关, N与P之间显著正相关。5)盐生植物体内总盐分、Na⁺和K⁺与N、N:P之间呈显著正相关关系, 与C、C:N、C:P之间呈显著负相关关系, 而K⁺和CO₃^2-与P之间却显著正相关。盐生植物体内盐分和养分在应对盐渍环境上存在一定的相互促进关系, 增施N肥有助于盐生植物对氯化物盐渍土的脱盐。这些发现为了解盐渍生境中盐生植物的生理生态适应及盐渍土的改良提供一定的理论依据。     

细胞药物的临床应用——细胞药物连载之五

近年来,细胞药物在基础研究领域的成果促进了临床应用。现在细胞药物治疗的疾病种类很多,包括神经系统疾病、自身免疫系统疾病、心血管系统疾病、血液系统疾病、消化系统疾病、下肢缺血、整形美容、抗衰老及抗肿瘤等,涉及的细胞主要有各种干细胞、软骨细胞、肝细胞、DC及CIK细胞等。国内外越来越多的机构和组织开展了细胞治疗的临床研究及应用。     

Theoretical and simulation studies on voltage-gated sodium channels

Voltage-gated sodium (Na_v) channels are indispensable membrane elements for the generation and propagation of electric signals in excitable cells. The successes in the crystallographic studies on prokaryotic Na_v channels in recent years greatly promote the mechanistic investigation of these proteins and their eukaryotic counterparts. In this paper, we mainly review the progress in computational studies, especially the simulation studies, on these proteins in the past years.     

Assessment of Phytotoxicity of Chromium in Flooded Soils using Embedded Selective Ion Exchange Resin Method

Chromium present in the forms of Cr(VI) or Cr(III) in soils. Since the toxicity and mobility of Cr(VI) are higher than those of Cr(III), it would be important to estimate soil Cr(VI) accurately in order to assess the phytotoxicity of Cr. Soil redox potential can influence the distribution of Cr between Cr(VI) and Cr(III) forms, and thus an in situ method which is not affected by the soil redox condition is needed for determining Cr(VI) availability in paddy fields. In this study, the Cu-saturated selective ion exchange resin (DOWEX M4159), serving as an infinite sink, was embedded in soils to extract available Cr(VI) from three representative saturated soils with different amounts of Cr(VI). The results suggested that Cr(VI) reduction occurred in the flooded soils, and the acid environment favored the adsorption and reduction of Cr(VI). There was a significant dose-response relationship between the soil resin-extractable Cr(VI) and the plant height of rice seedlings for test soils. The experimental results suggested that the embedded selective ion exchange resin method could be a suitable in situ method for assessing the phytotoxicity of Cr in flooded soils.     

Effects of counter-ion size on solid state structures of M(Nitrilotriacetate) complexes

The crystal structure of Na[Co(NC₆H₆O₆)] · H₂O is reported. The structure is compared to similar transition-metal nitrilotriacetate complexes containing different alkali cations and transition metals (Cu²⁺ and Zn²⁺). Inner-sphere coordination of the metals is similar, but the arrangement of counter-ions and water molecules in the unit cells vary with the size of the alkali cation.     

Cell elongation as an inseparable component of growth in terrestrial plants

The review is dedicated to the role of cell elongation in plant growth and morphogenesis. The ratios of cell division to elongation, cell competence for the initiation of elongation, main features of the metabolism of elongating cells, and physiological processes realizing elongation have been considered on the examples of seed germination and growth of roots, stems, and leaves. A special attention was paid to the vacuole as a specific feature of plant cells, pathways of its formation, and its role in maintenance of ion and water homeostasis in the elongating cell. The plant can modify its morphology according to changes in the environmental conditions via cell elongation.