The formulation of the culture medium for a Chinese hamster ovary (CHO) cell line has been investigated in terms of the simultaneous replacement of glucose and glutamine, the most commonly employed carbon and nitrogen sources, pursuing the objective of achieving a more efficient use of these compounds, simultaneously avoiding the accumulation of lactate and ammonium in the medium. The key factor in this process is the selection of compounds that are slowly metabolized. Among the different compounds studied, galactose and glutamate provide the best results, allowing support of cell growth with an optimal balance between nutrient uptake and cell requirements and the generation of minimal quantities of lactate and ammonium. The attained results also highlight the capacity of the cells to redistribute their metabolism as a response to the changes in medium composition.

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t‐PA‐producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t‐PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t‐PA production in continuous culture.

Nanomedicine plays an essential role in developing new therapies through novel drug delivery systems, diagnostic and imaging systems, vaccine development, antibacterial tools, and high-throughput screening. One of the most promising drug delivery systems are nanoparticles, which can be designed with various compositions, sizes, shapes, and surface modifications. These nanosystems have improved therapeutic profiles, increased bioavailability, and reduced the toxicity of the product they carry. However, the clinical translation of nanomedicines requires a thorough understanding of their properties to avoid problems with the most questioned aspect of nanosystems: safety. The particular physicochemical properties of nano-drugs lead to the need for additional safety, quality, and efficacy testing. Consequently, challenges arise during the physicochemical characterization, the production process, in vitro characterization, in vivo characterization, and the clinical stages of development of these biopharmaceuticals. The lack of a specific regulatory framework for nanoformulations has caused significant gaps in the requirements needed to be successful during their approval, especially with tests that demonstrate their safety and efficacy. Researchers face many difficulties in establishing evidence to extrapolate results from one level of development to another, for example, from an in vitro demonstration phase to an in vivo demonstration phase. Additional guidance is required to cover the particularities of this type of product, as some challenges in the regulatory framework do not allow for an accurate assessment of NPs with sufficient evidence of clinical success. This work aims to identify current regulatory issues during the implementation of nanoparticle assays and describe the major challenges that researchers have faced when exposing a new formulation. We further reflect on the current regulatory standards required for the approval of these biopharmaceuticals and the requirements demanded by the regulatory agencies. Our work will provide helpful information to improve the success of nanomedicines by compiling the challenges described in the literature that support the development of this novel encapsulation system. We propose a step-by-step approach through the different stages of the development of nanoformulations, from their design to the clinical stage, exemplifying the different challenges and the measures taken by the regulatory agencies to respond to these challenges.

Piscirickettsia salmonis is a fish bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry since its appearance in 1989. As this Gram-negative bacterium has been poorly characterized, relevant aspects of its life cycle, virulence and pathogenesis must be identified in order to properly design prophylactic procedures. This report provides evidence of the functional presence in P. salmonis of four genes homologous to those described for Dot/Icm Type IV Secretion Systems. The Dot/Icm System, the major virulence mechanism of phylogenetically related pathogens Legionella pneumophila and Coxiella burnetii, is responsible for their intracellular survival and multiplication, conditions that may also apply to P. salmonis. Our results demonstrate that the four P. salmonis dot/icm homologues (dotB, dotA, icmK and icmE) are expressed both during in vitro tissue culture cells infection and growing in cell-free media, suggestive of their putative constitutive expression. Additionally, as it happens in other referential bacterial systems, temporal acidification of cell-free media results in over expression of all four P. salmonis genes, a well-known strategy by which SSTIV-containing bacteria inhibit phagosome-lysosome fusion to survive. These findings are very important to understand the virulence mechanisms of P. salmonis in order to design new prophylactic alternatives to control the disease.