The Regulation of Biological and other Innovative Medicinal Products

This chapter discusses the phrase ‘biological medicinal product’, which is used to refer to products manufactured by biological or biotechnology means or to advanced therapy medicinal products. It examines biological medicinal products that are considered peptides or proteins and are made up of one or more linear sequences of amino acids. It also talks about the compliance of biological products with a minimum quality standard set by the British Pharmacopoeia or European Pharmacopoeia, which includes specific instructions for testing sterility, bacterial endotoxins, microbial limits, volume in container, uniformity of dosage units, and particulate matter. The chapter highlights biological products that are manufactured using recombinant cells and are extracted or made from unaltered tissues or blood that are purified in the same way as recombinant products. It explores the inherent heterogeneity of biological products as it comprises of a mixture of closely related molecules.

Cloning, Expression and Structural Modeling of the MlrA Protein from Novosphingobium sp. KKU25s for Microcystin Degradation

MlrA is a gene involved in the degradation of toxic cyanobacterial microcystins. This gene encodes microcystinase, mlrA, the 1st enzyme in the pathway that breaks down toxic cyanobacterial microcystins. In this study, primers were designed, and polymerase chain reaction (PCR) was performed to amplify the mlrA gene in Novosphincgobium sp. KKU25s. A PCR product of 752 base pairs was obtained. The nucleotide sequence of the mlrA gene of Novosphingobium sp. KKU25s was similar to that of Sphingomonas sp. ACM-3962 (98 % similarity). The mlrA gene of Novosphingobium sp. KKU25s was further cloned into the pGEM T-Easy plasmid to obtain the nucleotide sequence of the mlrA gene. The gene was also ligated into the pET32a plasmid for gene expression. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and verified using SDS-PAGE. The expressed protein was approximately 22 kilodaltons. The cell-free extract (CE) containing the crude protein from confirmed recombinant cells showed high activity in the biodegradation of [Dha7] MC-LR. [Dha7] MC-LR at an initial concentration of 30 mg L-1 and was completely biodegraded within 30 h. A distinct product derived from [Dha7] MC-LR appeared with a decrease in the [Dha7] MC-LR peak in the HPLC profile. The product (m/z 999.51) showed a molecular weight of 18, which is higher than that of native [Dha7] MC-LR (m/z 981.50), and was determined to be a linearized peptide fragment of [Dha7] MC-LR using LC-MS analysis. The 3-dimensional structure of microcystinase was predicted from the amino acid sequence deduced from the mlrA gene by the Swiss Model and Phyre2 programs. The structure contained a predicted alpha helix. The predicted 3-dimensional structure was also similar to that of a protein in the CAAX protease group. HIGHLIGHTS Research focused on characterization of microcystinase (MlrA) protein First research worked on the degradation of [Dha7] MC-LR by MlrA This work is useful for the applications aimed at the removal of MCs in freshwater environments GRAPHICAL ABSTRACT

Optimization of Multiparameters for Increased Yields of Cytochrome B5 in Bioreactors

The production of recombinant proteins is gaining increasing importance as the market requests high quality proteins for several applications. However, several process parameters affect both the growth of cells and product yields. This study uses high throughput systems and statistical methods to assess the influence of fermentation conditions in lab-scale bioreactors. Using this methodology, it was possible to find the best conditions to produce cytochrome b5 with recombinant cells of Escherichia coli. Using partial least squares, the height-to-diameter ratio of the bioreactor, aeration rate, and PID controller parameters were found to contribute significantly to the final biomass and cytochrome concentrations. Hence, we could use this information to fine-tune the process parameters, which increased cytochrome production and yield several-fold. Using aeration of 1 vvm, a bioreactor with a height-to-ratio of 2.4 and tuned PID parameters, a production of 72.72 mg/L of cytochrome b5 in the culture media, and a maximum of product to biomass yield of 24.97 mg/g could be achieved.

Analysis of the Innovation Trend in Cell-Free Synthetic Biology

Cell-free synthetic biology is a maturing field that aims to assemble biomolecular reactions outside cells for compelling applications in drug discovery, metabolic engineering, biomanufacturing, diagnostics, and education. Cell-free systems have several key features. They circumvent mechanisms that have evolved to facilitate species survival, bypass limitations on molecular transport across the cell wall, enable high-yielding and rapid synthesis of proteins without creating recombinant cells, and provide high tolerance towards toxic substrates or products. Here, we analyze ~750 published patents and ~2000 peer-reviewed manuscripts in the field of cell-free systems. Three hallmarks emerged. First, we found that both patent filings and manuscript publications per year are significantly increasing (five-fold and 1.5-fold over the last decade, respectively). Second, we observed that the innovation landscape has changed. Patent applications were dominated by Japan in the early 2000s before shifting to China and the USA in recent years. Finally, we discovered an increasing prevalence of biotechnology companies using cell-free systems. Our analysis has broad implications on the future development of cell-free synthetic biology for commercial and industrial applications.

The Schizophrenia Variant V1282F in SCN2A Causes Functional Impairment of NaV1.2

Neuropsychiatric disorders such as schizophrenia are challenging to treat due to the biological complexity of the disease and the lack of knowledge of the underlying pathophysiology. Whole exome and genome sequencing studies have identified disease-linked rare variants in patients with large effect size. Here, we functionally characterize the schizophrenia linked variant V1282F in SCN2A, encoding the sodium channel Nav1.2. This variant was introduced into isogenic lines of hiPSCs using CRISPR/CAS9 genome editing tools. hiPSCs were then differentiated into cortical neurons to understand how the variant and gene may be contributing to disease. We observed a significant (~25%) decrease in sodium current in the V1282F neurons compared to control neurons, suggesting the mutation is causing a loss-of-channel function. These results were supported by recordings in recombinant cells overexpressing either the mutant or wildtype Nav1.2, with the mutant channel having significantly (~75%) lower current amplitude than wildtype. We hypothesize that this phenotype may contribute to disease either through the direct loss of neuronal activity or through subsequent abnormal neurodevelopment.

Enhanced Extracellular Synthesis of Gold Nanoparticles by Soluble Extracts from Escherichia coli Transformed with Rhizobium tropici Phytochelatin Synthase Gene

Phytochelatins, the enzymatic products of phytochelatin synthase, play a principal role in protecting the plants from heavy metal and metalloid toxicity due to their ability to scavenge metal ions. In the present study, we investigated the capacity of soluble intracellular extracts from E. coli cells expressing R. tropici phytochelatin synthase to synthesize gold nanoparticle. We discovered that the reaction mediated by soluble extracts from the recombinant E. coli cells had a higher yield of gold nanoparticles, compared to that from the control cells. The compositional and morphological properties of the gold nanoparticles synthesized by the intracellular extracts from recombinant cells and control cells were similar. In addition, this extracellular nanoparticle synthesis method produced purer gold nanoparticles, avoiding the isolation of nanoparticles from cellular debris when whole cells are used to synthesize nanoparticles. Our results suggested that phytochelatins can improve the efficiency of gold nanoparticle synthesis mediated by bacterial soluble intracellular extracts, and the potential of extracellular nanoparticle synthesis platform for the production of nanoparticles in large quantity and pure form is worth further investigation.

Simultaneous quantification of mRNA and protein in single cells reveals post-transcriptional effects of genetic variation

Trans-acting DNA variants may specifically affect mRNA or protein levels of genes located throughout the genome. However, prior work compared trans-acting loci mapped in separate studies, many of which had limited statistical power. Here, we developed a CRISPR-based system for simultaneous quantification of mRNA and protein of a given gene via dual fluorescent reporters in single, live cells of the yeast Saccharomyces cerevisiae. In large populations of recombinant cells from a cross between two genetically divergent strains, we mapped 86 trans-acting loci affecting the expression of ten genes. Less than 20% of these loci had concordant effects on mRNA and protein of the same gene. Most loci influenced protein but not mRNA of a given gene. One locus harbored a premature stop variant in the YAK1 kinase gene that had specific effects on protein or mRNA of dozens of genes. These results demonstrate complex, post-transcriptional genetic effects on gene expression.

Elucidating the Efficacy of Vaccination against Vibriosis in Lates calcarifer Using Two Recombinant Protein Vaccines Containing the Outer Membrane Protein K (r-OmpK) of Vibrio alginolyticus and the DNA Chaperone J (r-DnaJ) of Vibrio harveyi

Recombinant cell vaccines expressing the OmpK and DnaJ of Vibrio were developed and subsequently, a vaccination efficacy trial was carried out on juvenile seabass (~5 cm; ~20 g). The fish were divided into 5 groups of 50 fish per group, kept in triplicate. Groups 1 and 2 were injected with 107 CFU/mL of the inactivated recombinant cells vaccines, the pET-32/LIC-OmpK and pET-32/LIC-DnaJ, respectively. Group 3 was similarly injected with 107 CFU/mL of inactivated E. coli BL21 (DE3), Group 4 with 107 CFU/mL of formalin killed whole cells V. harveyi, and Group 5 with PBS solution. Serum, mucus, and gut lavage were used to determine the antibody levels before all fish were challenged with V. harveyi, V. alginolyticus, and V. parahemolyticus, respectively on day 15 post-vaccination. There was significant increase in the serum and gut lavage antibody titers in the juvenile seabass vaccinated with r-OmpK vaccine. In addition, there was an up-regulation for TLR2, MyD88, and MHCI genes in the kidney and intestinal tissues of r-OmpK vaccinated fish. At the same time, r-OmpK triggered higher expression level of interleukin IL-10, IL-8, IL-1ß in the spleen, intestine, and kidney compared to r-DnaJ. Overall, r-OmpK and r-DnaJ triggered protection by curbing inflammation and strengthening the adaptive immune response. Vaccinated fish also demonstrated strong cross protection against heterologous of Vibrio isolates, the V. harveyi, V. alginolyticus, and V. parahaemolyticus. The fish vaccinated with r-OmpK protein were completely protected with a relative per cent of survival (RPS) of 90 percent against V. harveyi and 100 percent against V. alginolyticus and V. parahaemolyticus. A semi-quantitative PCR detection of Vibrio spp. from the seawater containing the seabass also revealed that vaccination resulted in reduction of pathogen shedding. In conclusion, our results suggest r-OmpK as a candidate vaccine molecule against multiple Vibrio strain to prevent vibriosis in marine fish.

Scavenger Receptor Class B Type I Is More Conducive to Hepatitis C Virus Invasion Compared with Low-Density Lipoprotein Receptor

Background: Hepatitis C virus is the major cause of chronic hepatitis which may deteriorate into liver cirrhosis or hepatocellular carcinoma. A number of studies have demonstrated that HCV cell entry is a complex multi-step process involving several cellular proteins, such as scavenger receptor class B type I (SR-BI), tetraspanin CD81, tight junction protein claudin-1 (CLDN-1) and occludin (OCLN). The low-density lipoprotein receptor (LDLR) is an important factor during the initial HCV particle-binding step, which interacts with the complex formed between the virus particle and the lipoprotein in the blood. However, the process of HCV early infection is not well-established, with many details remaining to be elucidated.This research aimed to study the early entry stage of HCV virus particles and the role of LDLR more effectively.Methods: Recombinant murine cell models of HCV infection in vitro was constructed, that expressed human HCV receptors, such as LDLR, CD81, SR BI, CLDN-1, and OCLN. These factors were also introduced to mice by hydrodynamic delivery to construct a humanized mouse model of HCV infection in vivo.Expression levels of the mRNA of HCV entry factors in recombinant cells were measured by qRT-PCR.Western blotting was used to determine whether the recombinant cells successfully expressed cellular proteins. HCV RNA was assayed by q-PCR following the incublation of HCVsd and HCVcc with the transgenice.Results: Transgenic murine cell lines and mice were developed successfully, and expressed four or five human HCV entry factors in tandem or individually, respectively. We found that all of these transgenic cells and mice were susceptible to HCV, and five entry factors (5EF) rendered higher infectivity. Additionally, we observed that four entry factors (4EF/hLDLR-) could facilitate abundant HCV entry, but four other factors (4EF/hSR-BI-) were less effective.Conclusions: Whether in vitro or in vivo, SR-BI is an essential factor in HCV invasion, and target cells and mice were more vulnerable to the virus in the presence of SR-BI than LDLR. These results suggested that SR-BI may be a potential drug target to inhibit HCV early infection, and the absence of LDLR could reduce the infectivity to the virus.