Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors

During the scale‐up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale‐up. Scale‐down simulators, which aim at replicating the large‐scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation mechanisms, which cells are using to adjust to rapidly fluctuating environmental conditions and can identify possible root causes for difficulties maintaining similar process performance at different scales. This understanding is of utmost importance in process validation. Additionally, these simulators also have the potential to be used for selecting cells, which are most robust when encountering changing extracellular conditions. The aim of this review is to summarize recent work in this interesting and promising area with the focus on mammalian bioprocesses, since microbial processes have been extensively reviewed.     

Simulations on the Primitive Electrolyte Environment of a High Charge-density Polyelectrolyte. A Sampling Problem and its Solution

Abstract

We show that the classical Metropolis Monte Carlo (MMC) algorithm converges very slowly when applied to the primitive electrolyte environment for a high charge-density polyelectrolyte. This slowness of convergence, which is due to the large density inhomogeneity around the polyelectrolyte, produces noticeable errors in the ion distribution functions for MMC runs of 1.3 × 10⁶ trial steps started from nonequilibrium distributions. We report that an algorithm which we call DSMC (for density-scaled Monte Carlo) overcomes this problem and provides relatively rapid convergence in this application. We suggest that DSMC should be well-suited for other Monte Carlo simulations on physical systems where large density inhomogeneities occur.     

Population heterogeneity and color stimulus heterogeneity in agent-based color categorization

Investigating the interactions between universal and culturally specific influences on color categorization across individuals and cultures has proven to be a challenge for human color categorization and naming research. The present article simulates the evolution of color lexicons to evaluate the role of two realistic constraints found in the human phenomenon: (i) heterogeneous observer populations and (ii) heterogeneous color stimuli. Such constraints, idealized and implemented as agent categorization and communication games, produce interesting and unexpected consequences for stable categorization solutions evolved and shared by agent populations. We find that the presence of a small fraction of color deficient agents in a population, or the presence of a “region of increased salience” in the color stimulus space, break rotational symmetry in population categorization solutions, and confine color category boundaries to a subset of available locations. Further, these heterogeneities, each in a different, predictable, way, might lead to a change of category number and size. In addition, the concurrent presence of both types of heterogeneity gives rise to novel constrained solutions which optimize the success rate of categorization and communication games. Implications of these agent-based results for psychological theories of color categorization and the evolution of color naming systems in human societies are discussed.     

Site inhomogeneity and exciton delocalization in the photosynthetic antenna

The influence of energy disorder on exciton states of molecular aggregates (the dimer and the circular aggregate) was analyzed. The dipole strength and inhomogeneous line shapes of exciton states were calculated by means of numerical diagonalization of Hamiltonian with diagonal energy disorder without intersite correlation. The disorder degree corresponding to destruction of coherent exciton states was estimated. The circular aggregates were treated as a model of light-harvesting antenna structures of photosynthetic bacteria. It was concluded that the site inhomogeneity typical for LH1 and LH2 complexes of purple bacteria cannot significantly influence the exciton delocalization over the whole antenna.Abbreviations BChl- bacteriochlorophyll - LH1 and LH2- core and peripheral light-harvesting complexes from purple bacteria - RC- reaction center     

A multisegmental cross-bridge kinetics model of the myofibril

Striated muscle is a mechanical system that develops force and generates power in serving vital activities in the body. Striated muscle is a complex biological system; a single mammalian muscle fibre contains up to hundred or even more myofibrils in parallel connected via an inter-myofibril filament network. In one single myofibril thousands of sarcomeres are lined up as a series of linear motors. We recently demonstrated that half-sarcomeres (hS) in a single myofibril operate non-uniformly. We outline a mathematical framework based on cross-bridge kinetics for the simulation of the force response and length change of individual hS in a myofibril. The model describes the muscle myofibril in contraction experiments under various conditions. The myofibril is modeled as a multisegmental mechanical system of hS models, which have active and viscoelastic properties. In the first approach, a two-state cross-bridge formalism relates the hS force to the chemical kinetics of ATP hydrolysis, as first described by Huxley [1957. Muscle structure and theories of contraction. Prog. Biophys. Mol. Biol. 7, 255-318]. Two possible types of biological variability are introduced and modeled. Numerical simulations of a myofibril composed of four to eight hS show a non-uniform hS length distribution and complex internal dynamics upon activation. We demonstrate that the steady-state approximation holds only in restricted time zones during activation. Simulations of myofibril contraction experiments that reproduce the classic steady-state force-length and force-velocity relationships, strictly constrained or “clamped” in either end-held isometric or isotonic contraction conditions, reveal a small but conspicuous effect of hS dynamics on force.     

Numerical investigation of thermal response of laser-irradiated biological tissue phantoms embedded with gold nanoshells

The work presented in this paper focuses on numerically investigating the thermal response of gold nanoshells-embedded biological tissue phantoms with potential applications into photo-thermal therapy wherein the interest is in destroying the cancerous cells with minimum damage to the surrounding healthy cells. The tissue phantom has been irradiated with a pico-second laser. Radiative transfer equation (RTE) has been employed to model the light-tissue interaction using discrete ordinate method (DOM). For determining the temperature distribution inside the tissue phantom, the RTE has been solved in combination with a generalized non-Fourier heat conduction model namely the dual phase lag bio-heat transfer model. The numerical code comprising the coupled RTE-bio-heat transfer equation, developed as a part of the current work, has been benchmarked against the experimental as well as the numerical results available in the literature. It has been demonstrated that the temperature of the optical inhomogeneity inside the biological tissue phantom embedded with gold nanoshells is relatively higher than that of the baseline case (no nanoshells) for the same laser power and operation time. The study clearly underlines the impact of nanoshell concentration and its size on the thermal response of the biological tissue sample. The comparative study concerned with the size and concentration of nanoshells showed that 60 nm nanoshells with concentration of 5×10¹⁵ mm⁻³ result into the temperature levels that are optimum for the irreversible destruction of cancer infected cells in the context of photo-thermal therapy. To the best of the knowledge of the authors, the present study is one of the first attempts to quantify the influence of gold nanoshells on the temperature distributions inside the biological tissue phantoms upon laser irradiation using the dual phase lag heat conduction model.     

The response of GS-NS0 myeloma cells to pH shifts and pH perturbations

The perceived sensitivity of animal cells to hydrodynamic shear has limited agitation and aeration at large-scale. This makes it difficult to ensure adequate mixing of the vessel contents and may lead to inhomogeneities in operational parameters such as temperature, dissolved oxygen concentration, and especially pH. The effect of pH shifts and pH perturbations on the cellular responses, in batch culture, of a GS-NS0 mouse myeloma cell line, expressing a recombinant antibody, was investigated. In addition, the effect of extreme pH on the structure of the purified antibody product was studied using isoelectric focusing. The fermentation pH value was shifted abruptly from pH 7.3 to pH values ranging from 6.5 to 9.0. Culture pH was maintained at this new value for the remainder of the fermentation. All pH shifts of above 0.2 units caused a transient increase in apoptosis. However, cultures shifted to pH values between 7.0 and 8.0 continued to grow and the apoptotic fraction returned to initial levels. Cultures shifted to pH values above pH 8.0 and below pH 7.0 did not recover resulting in culture death. For example, a shift to pH 8.5 caused accumulation of cells in the G(2)/M phase of the cell cycle followed by apoptotic death. After the pH shift, maximum specific growth rate was observed over the range pH 7.3 to 7.5 and maximum viable cell number was seen at pH 7.3. Maximum volumetric antibody production, resulting from increased culture longevity, was seen at pH 7.0. It was also observed that glucose consumption increased with increasing pH. In a separate set of experiments cells were subjected to a single pH perturbation ranging in duration from 0 to 600 minutes. Exposure of cells to a pH value greater than 8.5 for more than 10 minutes caused a decrease in the proportion of viable cells and induced a lag in cell growth. At very low pH (6.5) similar effects were seen, but only for extended perturbations (600 min). However, after recovery from the pH perturbation, growth, product secretion and metabolism all returned to original levels. Incubation of the antibody, at the range of pH values investigated, indicated no alterations in the structure of the antibody as determined by the isoelectric focusing pattern.     

Transverse interaction in DNA molecule

Interaction between nucleotides at a same site belonging to different strands is studied. This interaction is modelled by a Morse potential which depends on two parameters. We study a relationship between the parameters characterizing AT and CG pairs. We show that certain circumstances, i.e. certain values of these parameters, bring about a negligible influence of inhomogeneity on the solitonic dynamics.     

Raman spectroscopy characterization of antibody phases in serum

When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separation of mAbs in serum could guide lead optimization. However, serum contains numerous components, making it difficult to resolve mAb/serum mixtures at a scale amenable to analysis in a discovery setting. To address these challenges, a miniaturized assay was developed that combined confocal microscopy with Raman spectroscopy. The method was examined using CNTO607, a poorly-soluble anti-interleukin-13 human mAb, and CNTO3930, a soluble anti-respiratory syncytial virus humanized mAb. When CNTO607 was diluted into serum above 4.5 mg/mL, phase separation occurred, resulting in droplet formation. Raman spectra of droplet phases in mixtures included bands at 1240 and 1670 cm^?1, which are typical of mAb β-sheets, and lacked bands at 1270 and 1655 cm^?1, which are typical of α-helices. The continuous phases included bands at 1270 and 1655 cm^?1 and lacked those at 1240 and 1670 cm^?1. Therefore, CNTO607 appeared to be sequestered within the droplets, while albumin and other α-helix-forming serum proteins remained within the continuous phases. In contrast, CNTO3930 formed only one phase, and its Raman spectra contained bands at 1240, 1670, 1270 and 1655 cm,^?1 demonstrating homogeneous distribution of components. Our results indicate that this plate-based method utilizing confocal Raman spectroscopy to probe liquid-liquid phases in mAb/serum mixtures can provide a screen for phase separation of mAb candidates in a discovery setting.     

Raman spectroscopy characterization of antibody phases in serum

When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separation of mAbs in serum could guide lead optimization. However, serum contains numerous components, making it difficult to resolve mAb/serum mixtures at a scale amenable to analysis in a discovery setting. To address these challenges, a miniaturized assay was developed that combined confocal microscopy with Raman spectroscopy. The method was examined using CNTO607, a poorly-soluble anti-interleukin-13 human mAb, and CNTO3930, a soluble anti-respiratory syncytial virus humanized mAb. When CNTO607 was diluted into serum above 4.5 mg/mL, phase separation occurred, resulting in droplet formation. Raman spectra of droplet phases in mixtures included bands at 1240 and 1670 cm⁻¹, which are typical of mAb β-sheets, and lacked bands at 1270 and 1655 cm⁻¹, which are typical of α-helices. The continuous phases included bands at 1270 and 1655 cm⁻¹ and lacked those at 1240 and 1670 cm⁻¹. Therefore, CNTO607 appeared to be sequestered within the droplets, while albumin and other α-helix-forming serum proteins remained within the continuous phases. In contrast, CNTO3930 formed only one phase, and its Raman spectra contained bands at 1240, 1670, 1270 and 1655 cm,⁻¹ demonstrating homogeneous distribution of components. Our results indicate that this plate-based method utilizing confocal Raman spectroscopy to probe liquid-liquid phases in mAb/serum mixtures can provide a screen for phase separation of mAb candidates in a discovery setting.