Data on searching for synergy between alcohol and salt to produce more potent and environmentally benign gas hydrate inhibitors.

In order to systematically study the synergistic effect of gas hydrate inhibition with mixtures of methanol (MeOH) and magnesium chloride (MgCl2), the impact of these compounds on the thermodynamic stability of methane hydrate in the systems of CH4-MeOH-H2O, CH4-MgCl2-H2O, and CH4-MeOH-MgCl2-H2O was experimentally investigated. The pressure and temperature conditions of the three-phase vapor-aqueous solution-gas hydrate equilibrium were determined for these systems. The resulting dataset has 164 equilibrium points within the range of 234-289 K and 3-13 MPa. All equilibrium points were measured as the endpoint of methane hydrate dissociation during the heating stage. The phase boundaries of methane hydrate were identified for 8 systems with MeOH (up to 60 mass%), 5 MgCl2 solutions (up to 26.7 mass%), and 14 mixtures of both inhibitors. Most equilibrium points were measured using a ramp heating technique (0.1 K/h) under isochoric conditions when the fluids were stirred at 600 rpm. It was found that even a 0.5 K/h heating rate for the CH4-MgCl2-H2O system at low salt concentrations, along with all mixed aqueous solutions with methanol, gives results that do not differ from 0.1 K/h, considering the measurement uncertainties. Most measurements for the CH4-MgCl2-H2O system at high salt content were acquired using a step heating technique. The coefficients of the empirical equations approximating the equilibrium points for each inhibitor concentration were defined. The change in the slope parameter of the empirical equation was analyzed as a function of inhibitor content. Correlations that accurately describe the thermodynamic inhibition effect of methane hydrate with methanol and magnesium chloride on a mass% and mol% scale were obtained. The freezing temperatures of single and mixed aqueous solutions of methanol and magnesium chloride were determined experimentally to confirm the thermodynamic consistency of the methane hydrate equilibrium data.

Dataset for the dimethyl sulfoxide as a novel thermodynamic inhibitor of carbon dioxide hydrate formation.

The temperatures and pressures of the three-phase equilibrium V-Lw-H (gas - aqueous solution - gas hydrate) were measured in the CO2 - H2O - dimethyl sulfoxide (DMSO) system at concentrations of organic solute in the aqueous phase up to 50 mass%. Measurements of CO2 hydrate equilibrium conditions were carried out using a constant volume autoclave by continuous heating at a rate of 0.1 K/h with simultaneous stirring of fluids by a four-blade agitator at 600 rpm. The equilibrium temperature and pressure of CO2 hydrate were determined for the endpoint of the hydrate dissociation in each experiment. The CO2 gas fugacity was calculated by the equation of state for carbon dioxide for the measured points. The flow regime in the autoclave during the operation of the stirring system was characterized by calculating the Reynolds number using literature data on the viscosity and density of water and DMSO aqueous solutions. We employed regression analysis to approximate the dependences of equilibrium pressure (CO2 gas fugacity) on temperature by two- and three-parameter equations. For each measured point, the value of CO2 hydrate equilibrium temperature suppression ΔTh was computed. The dependences of this quantity on CO2 gas fugacity are considered for all DMSO concentrations. The coefficients of empirical correlation describing ΔTh as a function of the DMSO mass fraction in solution and the equilibrium gas pressure are determined. This article is a co-submission with a paper [1].

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells.

The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5' and 3' gene regulatory elements, and landing pad features on qP. We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with qP of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.

CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells.

The emergence of CRISPR/Cas9 system as a precise and affordable method for genome editing has prompted its rapid adoption for the targeted integration of transgenes in Chinese hamster ovary (CHO) cells. Targeted gene integration allows the generation of stable cell lines with a controlled and predictable behavior, which is an important feature for the rational design of cell factories aimed at the large-scale production of recombinant proteins. Here we present the protocol for CRISPR/Cas9-mediated integration of a gene expression cassette into a specific genomic locus in CHO cells using homology-directed DNA repair.

Minimizing Clonal Variation during Mammalian Cell Line Engineering for Improved Systems Biology Data Generation.

Mammalian cells are widely used to express genes for basic biology studies and biopharmaceuticals. Current methods for generation of engineered cell lines introduce high genomic and phenotypic diversity, which hamper studies of gene functions and discovery of novel cellular mechanisms. Here, we minimized clonal variation by integrating a landing pad for recombinase-mediated cassette exchange site-specifically into the genome of CHO cells using CRISPR and generated subclones expressing four different recombinant proteins. The subclones showed low clonal variation with high consistency in growth, transgene transcript levels and global transcriptional response to recombinant protein expression, enabling improved studies of the impact of transgenes on the host transcriptome. Little variation over time in subclone phenotypes and transcriptomes was observed when controlling environmental culture conditions. The platform enables robust comparative studies of genome engineered CHO cell lines and can be applied to other mammalian cells for diverse biological, biomedical and biotechnological applications.

Using Titer and Titer Normalized to Confluence Are Complementary Strategies for Obtaining Chinese Hamster Ovary Cell Lines with High Volumetric Productivity of Etanercept.

The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements.