In-silico media optimization for continuous cultures using genome scale metabolic networks: The case of CHO-K1.

The cell culture is the central piece of a biotechnological industrial process. It includes upstream (e.g. media preparation, fixed costs, etc.) and downstream steps (e.g. product purification, waste disposal, etc.). In the continuous mode of cell culture, a constant flow of fresh media replaces culture fluid until the system reaches a steady state. This steady state is the standard operation mode which, under very general conditions, is a function of the ratio between the cell density and the dilution rate and depends on the media supplied to the culture. To optimize the production process it is widely accepted that the concentration of the metabolites in this media should be carefully tuned. A poor media may not provide enough nutrients to the culture, while a media too rich in nutrients may be a waste of resources because, either the cells do not use all of the available nutrients, or worse, they over-consume them producing toxic byproducts. In this study, we show how an in-silico study of a genome scale metabolic network coupled to the dynamics of a chemostat could guide the strategy to optimize the media to be used in a continuous process. Given a known media we model the concentrations of the cells in a chemostat as a function of the dilution rate. Then, we cast the problem of optimizing the production process within a linear programming framework in which the goal is to minimize the cost of the media keeping fixed the cell concentration for a given dilution rate in the chemostat. We evaluate our results in two metabolic models: first a simplified model of mammalian cell metabolism, and then in a realistic genome-scale metabolic network of mammalian cells, the Chinese hamster ovary cell line. We explore the latter in more detail given specific meaning to the predictions of the concentrations of several metabolites.

Sodium acetate increases the productivity of HEK293 cells expressing the ECD-Her1 protein in batch cultures: experimental results and metabolic flux analysis.

Human Embryonic Kidney cells (HEK293) are a popular host for recombinant protein expression and production in the biotechnological industry. This has driven within both, the scientific and the engineering communities, the search for strategies to increase their protein productivity. The present work is inserted into this search exploring the impact of adding sodium acetate (NaAc) into a batch culture of HEK293 cells. We monitored, as a function of time, the cell density, many external metabolites, and the supernatant concentration of the heterologous extra-cellular domain ECD-Her1 protein, a protein used to produce a candidate prostate cancer vaccine. We observed that by adding different concentrations of NaAc (0, 4, 6 and 8 mM), the production of ECD-Her1 protein increases consistently with increasing concentration, whereas the carrying capacity of the medium decreases. To understand these results we exploited a combination of experimental and computational techniques. Metabolic Flux Analysis (MFA) was used to infer intracellular metabolic fluxes from the concentration of external metabolites. Moreover, we measured independently the extracellular acidification rate and oxygen consumption rate of the cells. Both approaches support the idea that the addition of NaAc to the culture has a significant impact on the metabolism of the HEK293 cells and that, if properly tuned, enhances the productivity of the heterologous ECD-Her1 protein.

HEK293 producing the extracellular domain HER1: Full datasets of continuous fermentation process and metabolites analysis.

The data for provide evidences of the multi steady state of the human cell line HEK 293 was obtained from 2 L bioreactor continuous culture. A HEK 293 cell line transfected to produce soluble HER1 receptor was used. The bioreactor was operated at three different dilution rates in sequential manner. Daily samples of culture broth were collected, a total of 85 samples were processed. Viable cell concentration and culture viability was addressing by trypan blue exclusion method using a hemocytometer. Heterologous HER1 supernatant concentration was quantified by a specific ELISA and the metabolites by mass spectrometry coupled to a liquid chromatography. The primary data were collected in excel files, where it was calculated the kinetic and other variables by using mass balance and mathematical principles. It was compared the steady states behavior each other's to find out the existence of steady states' multiplicity, taking into account the stationary phase with respect to the cell density (which means its coefficient of variation is less than 20 %). From the metabolic measurements by using Liquid Chromatography coupled to mass spectrometry (LC-MS), it was also built the data matrix with the specific rates of the 76 metabolites obtained. The data were processed and analyzed, using multivariate data asssnalysis (MVDA) to reduce the complexity and to find the main patterns present in the data. We describe also the full data of the metabolites not only for steady states but also in the time evolution, which could help others in terms of modeling and deep understanding of HEK293 metabolism, especially under different culture conditions.

A COVID-19 vaccine candidate composed of the SARS-CoV-2 RBD dimer and Neisseria meningitidis outer membrane vesicles.

SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. Vaccines seek to block this interaction by eliciting neutralizing antibodies, most of which are directed toward the RBD. Many protein subunit vaccines require powerful adjuvants to generate a potent antibody response. Here, we report on the use of a SARS-CoV-2 dimeric recombinant RBD combined with Neisseria meningitidis outer membrane vesicles (OMVs), adsorbed on alum, as a promising COVID-19 vaccine candidate. This formulation induces a potent and neutralizing immune response in laboratory animals, which is higher than that of the dimeric RBD alone adsorbed on alum. Sera of people vaccinated with this vaccine candidate, named Soberana01, show a high inhibition level of the RBD-ACE2 interaction using RBD mutants corresponding to SARS-CoV-2 variants of concern and wild-type expressed using the phage display technology. To our knowledge, this is the first time that the immunostimulation effect of N. meningitidis OMVs is evaluated in vaccine candidates against SARS-CoV-2.

SARS-CoV-2 RBD-Tetanus Toxoid Conjugate Vaccine Induces a Strong Neutralizing Immunity in Preclinical Studies.

Controlling the global COVID-19 pandemic depends, among other measures, on developing preventive vaccines at an unprecedented pace. Vaccines approved for use and those in development intend to elicit neutralizing antibodies to block viral sites binding to the host's cellular receptors. Virus infection is mediated by the spike glycoprotein trimer on the virion surface via its receptor binding domain (RBD). Antibody response to this domain is an important outcome of immunization and correlates well with viral neutralization. Here, we show that macromolecular constructs with recombinant RBD conjugated to tetanus toxoid (TT) induce a potent immune response in laboratory animals. Some advantages of immunization with RBD-TT conjugates include a predominant IgG immune response due to affinity maturation and long-term specific B-memory cells. These result demonstrate the potential of the conjugate COVID-19 vaccine candidates and enable their advance to clinical evaluation under the name SOBERANA02, paving the way for other antiviral conjugate vaccines.