Protein aggregation in salt solutions Kastelic, Miha; Kalyuzhnyi, Yurij V.; Hribar-Lee, Barbara ...
Proceedings of the National Academy of Sciences,
05/2015, Letnik:
112, Številka:
21
Journal Article
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Protein aggregation is broadly important in diseases and in formulations of biological drugs. Here, we develop a theoretical model for reversible protein–protein aggregation in salt solutions. We ...treat proteins as hard spheres having square-well-energy binding sites, using Wertheim’s thermodynamic perturbation theory. The necessary condition required for such modeling to be realistic is that proteins in solution during the experiment remain in their compact form. Within this limitation our model gives accurate liquid–liquid coexistence curves for lysozyme and Formula IIIa-crystallin solutions in respective buffers. It provides good fits to the cloud-point curves of lysozyme in buffer–salt mixtures as a function of the type and concentration of salt. It than predicts full coexistence curves, osmotic compressibilities, and second virial coefficients under such conditions. This treatment may also be relevant to protein crystallization.
Significance Protein aggregation is a problem in amyloid and other diseases, and it is a challenge when formulating solutions of biological drugs, such as monoclonal antibodies. The physical processes of aggregation, especially in salt solutions, are not well understood. We model a protein as having multiple binding sites to other proteins, leading to orientational variations, dependent on salt. With few parameters and with knowledge of the cloud-point temperatures as a function of added salt, the model gives good predictions for properties including the liquid–liquid coexistence curves, the second virial coefficients, and others for lysozyme and gamma-crystallin.
Chinese hamster ovary (CHO) epithelial cells are one of the most used therapeutic medical lines for the production of different biopharmaceutical drugs. They have a high consumption rate with a fast ...duplication cycle that makes them an ideal biological clone. The higher accumulated amounts of toxic intracellular intermediates may lead to lower organism viability, protein productivity and manufactured biosimilar, so a careful optimal balance of medium, bioreactor operational parameters and bioprocess is needed. A precise phenomenological knowledge of metabolism's chemical transformations can predict problems that may arise during batch, semi-continuous fed batch and continuous reactor operation. For a better detailed understanding (and relations), future performance optimization and scaling, mechanistic model systems have been built. In this specific work, the main metabolic pathways in mammalian structured CHO cultures are reviewed. It starts with organic biochemical background, controlling associated phenomena and kinetics, which govern the sustaining conversion routes of biology. Then, individual turnover paths are described, overviewing standard mathematical formulations that are commonly applied in engineering. These are the core of black box modeling, which relates the substrates/products in a simplified relationship manner. Moreover, metabolic flux analysis (MFA)/flux balance analysis (FBA), that are traditionally characterizing mechanisms, are presented to a larger portion extent. Finally, similarities are discussed, illustrating the approaches for their structural design. Stated variables' equations, employed for the description of the growth in the controllable environmental conditions of a vessel, the researched reaction series of proliferating dividing CHO population, joint with the values of maximal enzymatic activity, and solutions are outlined. Processes are listed in a way so that a reader can integrate the state-of-the-art. Our particular contribution is also denoted.
This study presents the theory for liquid–liquid phase separation for systems of molecules modeling monoclonal antibodies. Individual molecule is depicted as an assembly of seven hard spheres, ...organized to mimic the Y-shaped antibody. We consider the antibody–antibody interactions either through Fab, Fab′ (two Fab fragments may be different), or Fc domain. Interaction between these three domains of the molecule (hereafter denoted as A, B, and C, respectively) is modeled by a short-range square-well attraction. To obtain numerical results for the model under study, we adapt Wertheim’s thermodynamic perturbation theory. We use this model to calculate the liquid–liquid phase separation curve and the second virial coefficient B 2. Various interaction scenarios are examined to see how the strength of the site–site interactions and their range shape the coexistence curve. In the asymmetric case, where an attraction between two sites is favored and the interaction energies for the other sites kept constant, critical temperature first increases and than strongly decreases. Some more microscopic information, for example, the probability for the particular two sites to be connected, has been calculated. Analysis of the experimental liquid–liquid phase diagrams, obtained from literature, is presented. In addition, we calculate the second virial coefficient under conditions leading to the liquid–liquid phase separation and present this quantity on the graph B 2 versus protein concentration.
We analyze the experimentally determined phase diagram of a γD-βB1 crystallin mixture. Proteins are described as dumbbells decorated with attractive sites to allow inter-particle interaction. We use ...thermodynamic perturbation theory to calculate the free energy of such mixtures and, by applying equilibrium conditions, also the compositions and concentrations of the co-existing phases. Initially we fit the Tcloudversus packing fraction η measurements for a pure (x2 = 0) γD solution in 0.1 M phosphate buffer at pH = 7.0. Another piece of experimental data, used to fix the model parameters, is the isotherm x2vs. η at T = 268.5 K, at the same pH and salt content. We use the conventional Lorentz-Berthelot mixing rules to describe cross interactions. This enables us to determine: (i) model parameters for pure βB1 crystallin protein and to calculate; (ii) complete equilibrium surface (Tcloud-x2-η) for the crystallin mixtures. (iii) We present the results for several isotherms, including the tie-lines, as also the temperature-packing fraction curves. Good agreement with the available experimental data is obtained. An interesting result of these calculations is evidence of the coexistence of three phases. This domain appears for the region of temperatures just out of the experimental range studied so far. The input parameters, leading good description of experimental data, revealed a large difference between the numbers of the attractive sites for γD and βB1 proteins. This interesting result may be related to the fact that γD has a more than nine times smaller quadrupole moment than its partner in the mixture.
For biotechnological drugs, it is desirable to formulate antibody solutions with low viscosities. We go beyond previous colloid theories in treating protein–protein self-association of molecules that ...are antibody-shaped and flexible and have spatially specific binding sites. We consider interactions either through fragment antigen (Fab-Fab) or fragment crystallizable (Fab-Fc) binding. Wertheim's theory is adapted to compute the cluster-size distributions, viscosities, second virial coefficients, and Huggins coefficients, as functions of antibody concentration. We find that the aggregation properties of concentrated solutions can be anticipated from simpler-to-measure dilute solutions. A principal finding is that aggregation is controllable, in principle, through modifying the antibody itself, and not just the solution it is dissolved in. In particular: (i) monospecific antibodies having two identical Fab arms can form linear chains with intermediate viscosities. (ii) Bispecific antibodies having different Fab arms can, in some cases, only dimerize, having low viscosities. (iii) Arm-to-Fc binding allows for three binding partners, leading to networks and high viscosities.
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•We model the aggregation properties of antibodies in aqueous solutions.•The model molecules have a realistic shape and flexibility.•Wertheim's theory is adapted to analyse the measurements.•Bi-specific antibodies have lower viscosities than the monospecific ones.
We analyze the experimentally determined phase diagram of a γD-βB1 crystallin mixture. Proteins are described as dumbbells decorated with attractive sites to allow inter-particle interaction. We use ...thermodynamic perturbation theory to calculate the free energy of such mixtures and, by applying equilibrium conditions, also the compositions and concentrations of the co-existing phases. Initially we fit the
T
cloud
versus
packing fraction
η
measurements for a pure (
x
2
= 0) γD solution in 0.1 M phosphate buffer at pH = 7.0. Another piece of experimental data, used to fix the model parameters, is the isotherm
x
2
vs. η
at
T
= 268.5 K, at the same pH and salt content. We use the conventional Lorentz-Berthelot mixing rules to describe cross interactions. This enables us to determine: (i) model parameters for pure βB1 crystallin protein and to calculate; (ii) complete equilibrium surface (
T
cloud
-
x
2
-
η
) for the crystallin mixtures. (iii) We present the results for several isotherms, including the tie-lines, as also the temperature-packing fraction curves. Good agreement with the available experimental data is obtained. An interesting result of these calculations is evidence of the coexistence of three phases. This domain appears for the region of temperatures just out of the experimental range studied so far. The input parameters, leading good description of experimental data, revealed a large difference between the numbers of the attractive sites for γD and βB1 proteins. This interesting result may be related to the fact that γD has a more than nine times smaller quadrupole moment than its partner in the mixture.
Phase diagram of the mixture of β-γ lens crystallins can be modeled as a solution of patchy dumbbells. β and γ lens crystallins can be modeled as dumbbells decorated with attractive sites.
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•Biochemical reactions, constraints, and measurements form a simplified kinetic model.•Temporal alteration of viable cells (biomass) goes hand in hand with metabolites.•Reversible ...behavior of macro reactions accounts for diversity of mammalian cells.•Kinetic model describes cell phases of Chinese hamster ovary (CHO) cells.
Cellular biological environments are difficult to analyse on the first principle basis. In this modeling work, a novel approach is presented which transits from a broad mechanistic description of the individual metabolic pathways to a simplified macro-kinetic model. The latter is characterized within the concept of metabolic flux analysis and associated elementary modes, which obey the constraints of phenotype. Unlike the piecewise-defined kinetic expressions with a discrete separate treatment of different cell phases, the Monod equation rate law is extended herein, allowing for a dynamic reversal of macro-reactions, and thus, a uniform continuous functionality from growth to death cultivation periods. The enzymatic biochemical reactions of complete metabolic network are defined to satisfy the composition of mammalian culture structure which reflects on stoichiometry. This affords us an opportunity to handle the density of the viable cells (biomass) along with central metabolisms’ transformations, rather than as a decoupled constituent function, as habitually assumed in the literature. The methodology of the extracellular metabolite measurements for the Chinese hamster ovary (CHO) cells is confronted, confirming the applicability in multi-scale biologically-relevant systems. Temporal species’ behavior reveals inflection points that switch among stages. In further extensions metabolic models have a potential to forecast the outcomes of growth medium perturbations, feeding protocols, and process parameters in order to facilitate the productivity and quality of biosimilars in biotechnology.
Experimental and modeling work, described in this article, is focused on the metabolic pathway of Chinese hamster ovary (CHO) cells, which are the preferred expression system for monoclonal antibody ...protein production. CHO cells are one of the primary hosts for monoclonal antibodies production, which have extensive applications in multiple fields like biochemistry, biology and medicine. Here, an approach to explain cellular metabolism with in silico modeling of a microkinetic reaction network is presented and validated with unique experimental results. Experimental data of 25 different fed‐batch bioprocesses included the variation of multiple process parameters, such as pH, agitation speed, oxygen and CO2 content, and dissolved oxygen. A total of 151 metabolites were involved in our proposed metabolic network, which consisted of 132 chemical reactions that describe the reaction pathways, and include 25 reactions describing N‐glycosylation and additional reactions for the accumulation of the produced glycoforms. Additional eight reactions are considered for accumulation of the N‐glycosylation products in the extracellular environment and one reaction to correlate cell degradation. The following pathways were considered: glycolysis, pentose phosphate pathway, nucleotide synthesis, tricarboxylic acid cycle, lipid synthesis, protein synthesis, biomass production, anaplerotic reactions, and membrane transport. With the applied modeling procedure, different operational scenarios and fed‐batch techniques can be tested.
Experimental and modeling work, described in this article, is focused on the metabolic pathway of Chinese hamster ovary cells, which are the preferred expression system for monoclonal antibody protein production. Here, an approach to explain cellular metabolism with in silico modeling of a microkinetic reaction network is presented and validated with unique experimental results.
Managing information technology services is becoming an increasingly difficult task. To support the management of IT services, different standards and methodologies have been developed. ITIL (short ...for IT Infrastructure Library) is the most commonly used best practice approach to effective IT Service Management to date. ITIL focuses primarily on what to do in order to ensure value of IT services, but it does not explain how to achieve this effectively. This shortcoming can be overcome by complementing the framework with other quality approaches to service management. In this context several methodologies are mentioned including the use of Six Sigma (6s) methodology. The statistical nature of the Six Sigma methodology enables us to analyze the vast amount of data gathered from the field of IT. Only after these value-based metrics are obtained can the overall health of the IT service be determined and the necessary improvement measures made. The aim of this paper is to analyze in detail the two approaches. We will establish a common link between them, with it the opportunity to complement ITIL with the Six Sigma methodology, and consequently set foundations for introduction of necessary measurable changes.
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