Basic Variants Research Articles

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics.

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

Differential evolution and particle swarm optimization against COVID-19

COVID-19 disease, which highly affected global life in 2020, led to a rapid scientific response. Versatile optimization methods found their application in scientific studies related to COVID-19 pandemic. Differential Evolution (DE) and Particle Swarm Optimization (PSO) are two metaheuristics that for over two decades have been widely researched and used in various fields of science. In this paper a survey of DE and PSO applications for problems related with COVID-19 pandemic that were rapidly published in 2020 is presented from two different points of view: 1. practitioners seeking the appropriate method to solve particular problem, 2. experts in metaheuristics that are interested in methodological details, inter comparisons between different methods, and the ways for improvement. The effectiveness and popularity of DE and PSO is analyzed in the context of other metaheuristics used against COVID-19. It is found that in COVID-19 related studies: 1. DE and PSO are most frequently used for calibration of epidemiological models and image-based classification of patients or symptoms, but applications are versatile, even interconnecting the pandemic and humanities; 2. reporting on DE or PSO methodological details is often scarce, and the choices made are not necessarily appropriate for the particular algorithm or problem; 3. mainly the basic variants of DE and PSO that were proposed in the late XX century are applied, and research performed in recent two decades is rather ignored; 4. the number of citations and the availability of codes in various programming languages seems to be the main factors for choosing metaheuristics that are finally used.

Evaluation of the energy-efficient operation of a two-level air-jet screen with coaxial counter jets with ejection feeding in combination with rotating flows

The use of local exhaust ventilation systems continues to be the most reliable way to capture pollutants in the technological processes of various industries. The main purpose of the application of local exhaust ventilation systems is the effective capture of pollutants in the places of their formation. Improving the efficiency of local exhausts and, accordingly, reducing the load of general ventilation can be achieved through the use of air screens. For baths filled with liquids most often used activated on-board suction pumps that operate on the scheme of "jet-suction", which are structurally located on the same axis of the supply nozzle and slotted drain. According to this principle of operation, various activated extractors require air exchange in large volumes with significant energy consumption and at the same time with insufficiently high sanitary and hygienic indicators. In order to increase the efficiency of the local ventilation system, especially for tanks with large open surfaces, fundamentally new aerodynamic schemes are required, the feature of which is the formation of dynamic screens with inflow and rotation flows. On the basis of analytical research the basic possible variants of the device of the two-level air-jet screen of an industrial bath by drawing up balance equations for achievement of the maximum efficiency factor η are considered. The analysis of theories of interactions of inflow jets, suction flares and convective flows from liquid surfaces is carried out. The aerodynamic problem of a flat turbulent flow is considered, in which inflow jets interact with a 180 ° expansion, forming a two-level closed rotating system with concentrated flow, which is affected by convective flows with harmful substances from an industrial bath. The assumptions and calculations confirmed the fundamental possibility of using a system of two-level air-jet screens for large baths.

Objectivation of the Ecological and Economic Losses from Solid Domestic Waste at the Heating Enterprises

Abstract The aim of this research is to study theoretical and practical aspects of the ecological and economic losses from the use of solid domestic waste (SDW) as energy resources in the heat power industry of Ukraine. The methodical approaches to evaluating the ecological and economic losses caused by solid domestic waste (SDW) comprise: the developed algorithm, which evaluates the ecological and economic losses in the SDW use as fuel and energy resources in comparison with basic and project variant; the investigated morphological composition of SDW in the Ukrainian regions, on the basis of which there is proposed a matrix for further calculations of the ecological and economic loss from atmospheric pollution as a result of the energy-intensive SDW combustion at the power plants by the Ukrainian regions. The efficiency of using SDW as secondary energy resources, which essentially depends on the conventional energy resources combustion, is proved. According to the chemical and morphological composition of SDW, the average amount of harmful substances by their element constituents of SDW is determined. The economic loss from the combusting 1 ton of SDW as energy resources is estimated. Reasonability of using SDW as energy resources, based on the optimal ratio between conventional resources and energy-intensive SDW through minimizing total production costs and possible ecological and economic loss, is grounded. It is proved that while estimating the ecological and economic losses, it is necessary to consider the SDW morphological composition and regional specific features regarding the location of heat and power enterprises and organized storage landfills. It is grounded that the obtained estimates of the ecological and economic losses may be used for identifying the ecological and economic evaluation of the SDW efficiency use in the heat power industry at the regional level.

SEDENTARY BURIALS ON THE SOUTHERN RUS’ TERRITORY

The article is devoted to the burials of the X—XI centuries, where the deceased were found in a sitting position. Despite the fact that this custom itself is undoubtedly of Norman origin, the issue of ethnic and social attribution of most complexes from the territory of Southern Rus’ has not received appropriate attention among the researchers. Information was collected on 17 Southern Rus’ burials, which, according to some researchers, were carried out in a sitting position. However, it is not always possible to establish the posture of the deceased with complete confidence. The basic variants of mutual arrangement of bone remains are allocated and on their basis the most widespread ways of giving to a body a sitting position are reconstructed.
 Both by the social composition of the buried and chronologically, the burial complexes can be divided into two separate groups. The first group includes rich burials in chamber tombs. All of them are dated by the X century and are located in the necropolises of Kyiv and Shestovytsia, two points of the highest concentration of the Scandinavian ethnic component in Southern Rus’. A wide range of objects of Scandinavian origin was found in the toms, there are burials with weapons, slaves and horses killed. Undoubtedly, the dead belonged to the military-trade elite. In combination with chamber tombs, a sitting posture can be an indicator of the Scandinavian origin of the dead.
 Sedentary burials of the second group are located in the peripheral areas of the Southern Rus’ and belong to the Christian era (approximately — the end of the X—XI centuries). Burials are carried out at the level of the day surface, are marked by rather «poor» inventory and, as a rule, do not have burial structures. They can be associated with the ordinary Slavic population and its efforts to recreate certain prestigious features of the funeral rites of the previous period. Probably, the sedentary position symbolized a dominant status in society, and in the funeral rite carried only a social, not a religious stress.

Effect of vitamins and metal ions on productivity and charge heterogeneity of IgG1 expressed in CHO cells.

Recombinant monoclonal antibodies have emerged as the most successful modality of biotherapeutics. They are primarily expressed in Chinese Hamster Ovary (CHO) cells. It is well known that post-translational modifications (PTM) contribute significantly to heterogeneity with respect to charge, glycosylation, and size. These attributes in turn impact stability, pharmacokinetics, and pharmacodynamics of the biotherapeutic product. Cell culture media components are known to significantly contribute to both cellular productivity as well as post-translational modifications. Thus, it is highly desirable to understand how media components affect product quality. This study aims to explore the impact of vitamins and metal ions on protein expression and post-translational modifications specifically charge heterogeneity. Biotin, choline chloride, D-calcium pantothenate, folic acid, pyridoxine hydrochloride, thiamine hydrochloride vitamins and Fe, Cu, Mg, Co, Zn, Mn, Ni metal ions were examined in this study. The results indicate that pyridoxine enhances productivity while Zn, Cu, Fe, Mn, and biotin impact charge heterogeneity. While, Fe, Mn and Ni enhance production of the acidic variants, Cu and biotin inhibit it. Zn reduces formation of basic variants while biotin enhances it. The results from this investigation could be used for process control so as to get consistent charge variant profile, in particular for biosimilars.

Iterative-deepening Bidirectional Heuristic Search with Restricted Memory

The field of bidirectional heuristic search has recently seen great advances. However, the subject of memory-restricted bidirectional search has not received recent attention. In this paper we introduce a general iterative deepening bidirectional heuristic search algorithm (IDBiHS) that searches simultaneously in both directions while controlling the meeting point of the search frontiers. First, we present the basic variant of IDBiHS, whose memory is linear in the search depth. We then add improvements that exploit consistency and front-to-front heuristics. Next, we move to the case where a fixed amount of memory is available to store nodes during the search and develop two variants of IDBiHS: (1) A*+IDBiHS, that starts with A* and moves to IDBiHS as soon as memory is exhausted. (2) A variant that stores partial forward frontiers until memory is exhausted and then tries to match each of them from the backward side. Finally, we experimentally compare the new algorithms to existing unidirectional and bidirectional ones. In many cases our new algorithms outperform previous ones in both node expansions and time.

Improvement of analytical results quality in neuroscience – Good Methodology Practice in the acetylcholine determination

A new point of view on ensuring the best quality of analytical results has been presented. We proposed the strategy called Good Methodology Practice leading to the improvement of the quality of analytical results and assessment of interference effects. Four calibration methods: External Calibration, Standard Addition Method, Integrated Calibration Method in the version of Complementary Dilution Method and basic variant of Integrated Calibration Method were tested in terms of acetylcholine determination. Acetylcholine is an important neurotransmitter that plays a crucial role i.e. in memory and movement. The determination of acetylcholine in mice brain was carried out with the use of microdialysis in freely moving animals followed by high-performance liquid chromatograph with electrochemical detection. The comparison of obtained results calculated in various ways allowed an effective assessment and compensation of systematic errors as well as the improvement of the accuracy of analytical results. The most suitable method was chosen.

Freeze thaw and lyophilization induced alteration in mAb therapeutics: Trastuzumab as a case study

Long-term stability of therapeutic monoclonal antibody (mAb) products is necessary for their successful commercialization. Freeze-thaw (F/T) operations are often performed for a mAb product during processing, storage and distribution. Lyophilization (Lyo) is another unit operation that is commonly used for drug product manufacturing of mAbs. This paper aims to explore the impact of these operations on structure and function of a mAb therapeutic, as well as of biosimilars. Trastuzumab innovator and its five biosimilars were analysed for aggregation, charge heterogeneity, secondary structure, binding kinetics, and potency after each freeze-thaw and lyophilization cycle. It is observed that both F/T and Lyo induce protein aggregation, which in turn causes perturbations in the biological potency of the mAb therapeutic. The average value of the percentage of aggregation increased from 0.6 % (week 1) to 5.3 % (week 10) in F/T study and from 0.8 % (week 1) to 10.1 % (week 10) in Lyo study. The acidic pool increased from 26.5 % (week 1) to 44.4 % (week 10) and the basic variants from 13.9 % (week 1) to 24.0 % (week 10) in F/T study. Similarly, acidic pool increased from 27.1 % (week 1) to 42.0 % (week 10) and basic variants from 14.8 % (week 1) to 24.4 % (week 10) in Lyo study. The average percentage of beta-sheet increased from 58.4 % (week 1) to 60.9 % (week 10) in F/T study and from 59.7 % (week 1) to 72.6 % (week 10) in Lyo study. Lower binding affinity was found in week 7 as compared to week 1 in Lyo study whereas no change in binding affinity was observed in the F/T study. The average potency value gradually decreased from 0.97IU/ ml (week 1) to 0.75IU/ ml (week 10) in F/T study and from 1.0IU/ ml (week 1) to 0.66IU/ ml (week 10) in Lyo study. Results indicate that lyophilization has a bigger impact on binding affinity than freeze thaw and as expected, the impact was comparable across the innovator and biosimilar products.

Some aspects of the theory of errors of doctors

The development of clinical thinking and the improvement of medical care largely depend on doctors and society’s attitude to medical errors. A balanced, rational perspective to medical errors is critical with an understanding of the main aspects of medication errors. The paper presents two important aspects of the problem of medical errors - their probability and, in a sense, their routine, as well as the role of collective relationships in shaping an objective attitude of doctors to errors. The article argues that the correct action of a doctor does not mean that they are unmistakable. The factors that determine the complexity of medicine are listed. The actual and ambiguous effectiveness of the medicine is considered. It is argued that the development of evidence-based medicine is evidence of the relativity of knowledge in medicine. The basic variants of the relationship of doctors in the collective are given. The role of collegiality in improving the professional experience and quality of medical care is specified. It has been shown that the absence of objective criteria of guilt or innocence of a doctor in error exacerbates the problem of errors. It emphasizes the flaw of equating all errors with misconduct and the unilateralism of such an approach in preventing mistakes. A vicious circle is presented, where the traditionally negative attitude towards doctors who made a mistake ultimately leads to concealment of errors, a decrease in the number and quality of error analysis, stagnation in the development of clinical thinking, an increase in the number of errors and, accordingly, the legal tightening of demand for medical errors.

Imiona chrześcijańskie w Synodyku nowogrodzkim z końca XIV–XV wieku

The article contains an analysis of Christian names selected from the oldest synodicon written in the Lisitsky monastery near Veliky Novgorod. The analysis comprises 3190 names of laypeople, amongst which are 208 male names and 58 female names. The author discusses the group of given names (basic forms, their phonetic, morphological variants and frequency, as well as rarely used names that occurred in the synodicon).

Exon-1 skipping and intron-1 retaining by alternative splicing of the c-KIT gene encodes a novel splice variant in the skin of Merino sheep (Ovis aries).

c-KIT, a type III receptor protein tyrosine kinase, plays an essential role in melanocyte development, migration, and survival. Mutations within the c-KIT gene are previously shown to cause the white coat color phenotypes in pigs, mice, goats, and humans. However, up so far, the splicing isoform(s), genomic architecture of c-KIT have not been characterized well in merino sheep. Reverse transcriptase (RT)-PCR analysis with molecular prediction identified two basic splice variants: Transcript Variant-1, 2 for 12bp insertion coding sequences (CDS) corresponding to the four amino acids 'GNSK', respectively. Using 5' RACE, here we report for the first time a novel c-KIT 'Transcript Variant-3' from the skin of merino sheep by comparative genome analyses at exon(1)-intron(1)-exon(2) boundaries. In contrast, a single product of 795bp was characterized by 3' RACE. We also demonstrated that the c-KIT gene expression at the transcript level is not mediated via an intron-9 splicing event. Overall, beyond what was observed in other mammals, our data provide novel insights into the molecular structure of the c-KIT gene in sheep.

Characterization and monitoring of charge variants of a recombinant monoclonal antibody using microfluidic capillary electrophoresis-mass spectrometry

Rigorous characterization of biotherapeutics, and monoclonal antibodies in particular, is a challenging task in terms of ensuring safety, efficacy, and potency of a therapeutic agent because of structural heterogeneity during cell culture, purification and storage. In this work, we used microfluidic capillary electrophoresis-mass spectrometry to analyze intact monoclonal antibody and assess the root cause of increases in acidic and basic variants under stress at high temperature. The antibody was analyzed at multiple levels, including its intact state under native conditions, and subunit and peptide levels. The normal and degraded antibodies at different time points were characterized and compared with each other. We concluded that the basic variants in the unstressed sample were produced C-terminal amidation, while the acidic variants were produced by deamidation. In stressed samples, change in the acidic and main peaks were caused by deamidation, and changes in the basic peaks were caused by both deamidation and oxidation. These results demonstrate that microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a powerful direct and generic tool for separation and identification of charge heterogeneity of biotherapeutics.

Multi-layer Gaussian-based multi-configuration time-dependent Hartree (ML-GMCTDH) simulations of ultrafast charge separation in a donor-acceptor complex.

We report on first applications of the Multi-Layer Gaussian-based Multi-Configuration Time-Dependent Hartree (ML-GMCTDH) method [Römer et al., J. Chem. Phys. 138, 064106 (2013)] beyond its basic two-layer variant. The ML-GMCTDH scheme provides an embedding of a variationally evolving Gaussian wavepacket basis into a hierarchical tensor representation of the wavefunction. A first-principles parameterized model Hamiltonian for ultrafast non-adiabatic dynamics in an oligothiophene-fullerene charge transfer complex is employed, relying on a two-state linear vibronic coupling model that combines a distribution of tuning type modes with an intermolecular coordinate that also modulates the electronic coupling. Efficient ML-GMCTDH simulations are carried out for up to 300 vibrational modes using an implementation within the QUANTICS program. Excellent agreement with reference ML-MCTDH calculations is obtained.

СТРУКТУРИРОВАНИЕ СОДЕРЖАНИЯ ВАРИАТИВНОЙ ИНОЯЗЫЧНОЙ ПОДГОТОВКИ В ВУЗЕ

The article deals with the issues of structuring the variative foreign language training content. Variative foreign language training consists of a basic mandatory part for all students, which implements the requirements of Federal state educational standards at a minimum level and variation part. The variation part, represented by educational variants, is designed to meet the cognitive needs of students and take into account personal characteristics that affect the acquisition of a foreign language at the University. It is shown that the system approach is the basis for structuring the content. Professionally oriented foreign language training in higher education is considered as part of the higher education system, is subject to general goals, is regulated by the standard, program, and includes the levels of higher education: bachelor's, master's, specialty, and postgraduate. Disciplines related to foreign language acquisition are focused on the formation of foreign language communication skills, and also allow forming flexible skills that are necessary by specialists in different areas of professional activity today and are increasingly in the future. It is noted that the system approach regulates the construction of variative training parts in the aggregate, due to their interaction. The core of the system is the basic and variation variants availability. The framework of the system is the educational standards requirements, the educational program data, which sets the terms of the course development, the number of hours for classroom and self-study work, the form of intermediate and final control. The system integrity is manifested in the presence of horizontal and vertical links between the educational process elements. Horizontal links are located in the relationship between the basic and variation parts at each education level. Vertical links are found between the basic and additional parts of all levels on the basis of content and technology continuity. Continuity is understood as taking into account the individual level of educational achievements and reliance on the studied material. The results led to structure the variative foreign language training content in "Animal husbandry", to identify the basic and variation parts for the growth and development of foreign language skills and orientation training to suit individual needs and students’ characteristics.

The Analysis of Small Investors’ Demands on a Thermal Insulation System for a Family House: A Case Study

This study deals with small investors’ demands on thermal insulation systems when choosing the most suitable solution for a family house. By 2050, seventy percent of current buildings, including residential buildings, are still expected to be in operation. To reach carbon neutrality, it is necessary to reduce operational energy consumption and thus reduce the related cost of building operations and the cost of the life cycle of buildings. One solution is to adapt envelopes of buildings by proper insulation solutions. To choose an optimal thermal insulation system that will reduce energy consumption of building, it is necessary to consider the environmental cost of insulation materials in addition to the construction cost of the materials. The environmental cost of a material depends on the carbon footprint from the initial origin of the material. This study presents the results of a multi-criteria decision-making analysis, where five different contractors set the evaluation criteria for selection of the optimal thermal insulation system. In their decision-making, they involved the requirements of small investors. The most common requirements were selected: the construction cost, the construction time (represented by the total man-hours), the thermal conductivity coefficient, the diffusion resistance factor, and the reaction to fire. The confidences of the criteria were then determined with the help of the pairwise comparison method. This was followed by multi-criteria decision-making using the method of index coefficients, also known as the method of basic variant. The multi-criteria decision-making included thermal insulation systems based on polystyrene, mineral wool, thermal insulation plaster, and aerogels’ nanotechnology. As a result, it was concluded that, currently, in Slovakia, small investors emphasize the cost of material and the coefficient of thermal conductivity and they do not care as much about the carbon footprint of the material manufacturing, the importance of which is mentioned in this study.

A Survey of Nature-Inspired Computing

Nature-inspired computing is a type of human-designed computing motivated by nature, which is based on the employ of paradigms, mechanisms, and principles underlying natural systems. In this article, a versatile and vigorous bio-inspired branch of natural computing, named membrane computing is discussed. This computing paradigm is aroused by the internal membrane function and the structure of biological cells. We first introduce some basic concepts and formalisms of membrane computing, and then some basic types or variants of P systems (also named membrane systems ) are presented. The state-of-the-art computability theory and a pioneering computational complexity theory are presented with P system frameworks and numerous solutions to hard computational problems (especially NP -complete problems) via P systems with membrane division are reported. Finally, a number of applications and open problems of P systems are briefly described.

LC\u2013MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.

C-terminal lysine clipping of IgG1: impact on binding to human FcγRIIIa and neonatal Fc receptors

Monoclonal antibodies (mAbs) display numerous structural attributes, some of them may impact their safety and/or efficacy profiles. C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants. If Fc-glycosylation has been by far the most documented critical quality attribute (CQA), the potential impacts of mAb C-terminal lysine content is far less reported, particularly on the ability of these basic variants to bind human Fc receptors. To address this question, three charge variant species having zero (K0), one (K1) and two (K2) C-terminal lysine(s) were isolated with high purity from an in-house human IgG1 by preparative strong-cation exchange (SCX) chromatography. A comprehensive biophysical characterization of these three fractions was undertaken, demonstrating their high similarity in terms of structural homogeneity, with a particular attention paid on their respective N-glycosylation profiles. The binding affinity of the fractions to human FcγRIIIa-Val176 was assessed both by affinity chromatography and surface plasmon resonance (SPR), and to human neonatal Fc receptor (FcRn) by affinity chromatography. Results demonstrate that the three charge variants did not show any significant binding difference for the two tested human Fc receptors, translating certainly to comparable biological properties. As a consequence, C-terminal lysine clipping of the present therapeutic IgG1 should not impact both FcRn-dependent pharmacokinetic profiles and FcγRIIIa-driven cytotoxic activities. The methods used in this study can be widely applied to other IgG1 to define criticality of the C-terminal lysine clipping as a CQA.

Mental Disorders in Patients at Geriatrician Attendance : Basic Variants and Influence on Social Functioning (The Experience in Russia)

Data exists showing a significant prevalence of mental disorders in the growing elderly population, but indices have a fairly wide range. This study’s aim was to analyze the frequency and variants of such disorders of the elderly in geriatrician attendance and their influence on social functioning.