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EMA 259
Well - updated by authors from the previous issue, we learn about personalized medicine and xenotransplantation, and we are proud to introduce new contributors who tell us about nanocarriers as future drug delivery systems, ultra-high-throughput screening for accelerated drug discovery, and transgenic plants as future green factories. Woerdenbag University of Groningen Pharmaceutical Technology and Biopharmacy Department Antonius Deusinglaan 1 9713 AV Groningen The Netherlands.
Part One
Concepts and Methods for Recombinant Drug Production
Pharmaceutical Biotechnology and Industrial
Synthetic biology is still in its infancy and has not been exposed to wide use in the pharmaceutical laboratories [16]. Two examples can illustrate the potential of metabolic engineering and synthetic biology to influence bioprocesses in the future.
Prokaryotic Cells in Biotech Production
The efficiency of translation initiation also depends on the secondary structure on the RBS of the mRNA. Today, one of the most commonly used techniques is the expression of the proinsulin precursor in E.
Mammalian Cells in Biotech Production
In our opinion, the opportunities to design “the” ideal mammalian host cell lines for the needs of industry (systems biology) have not yet been addressed today and may not be feasible in the near future, as “ the CHO genome” and “CHO biology” do not exist. This selection scheme, slightly modified when using other selection markers, remains one of the standard methods to establish stably transfected CHO cell lines for the production of recombinant therapeutic proteins. In this way, high-yielding cell lines with capacities to produce 1 – 3 g/l (after further development work) have been identified (De Jesus, unpublished data).
The level of MTX-specific fluorescence is expected to correlate with the level of the recombinant protein of interest. Once clonal cell lines are established, they must be characterized for "stability" of recombinant protein production. Gene silencing is hypothesized to be influenced by environmental DNA in the vicinity of integrated GOI sequences.
Rapid gene silencing occurs in about half of the cell lines within days of releasing the cells from selective pressure. Production of recombinant proteins by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. production of recombinant proteins by suspension - adapted HEK293 - EBNA cells.
Biopharmaceuticals from Plants
Regarding recombinant protein production, plastids are truly exceptional because they allow protein accumulation up to 70% of the total protein (as in the case of antimicrobial peptides [8). Even the inoculation process of plants can be simplified there. The surface parts of the plant (Nicotiana benthamiana) are immersed in an Agrobacteria solution and a vacuum is applied. Cell-to-cell movement of viral replicons generalizes the infection and results in uniform expression of the transgene throughout the plant parts.
Protein glycosylation can also be different, as illustrated by the production of the enzyme Phytase from Aspergillus niger [13], which showed different glycosylation patterns when produced in either the leaves or seeds of Oryza sativa (rice). Since the identification of the encoding gene and its expression in mammalian cell cultures (Chinese hamster ovary (CHO) cells), sufficient amounts of protein can be provided for the effective enzyme replacement therapy of Gaucher's disease. In addition to the obvious advantage of the unique purification scheme, the production of recombinant pharmaceuticals in safflower has another advantage.
In addition to the traditional mammalian cell cultures for the production of the surface proteins hemagglutinin and neuraminidase from influenza viruses, plant-based systems are also being developed. As mentioned at the outset, a transient expression system still has the disadvantage that manual inoculation of the plant with the vector (either viruses or agrobacteria) must be performed.
Production of Biopharmaceuticals in Transgenic Animals
Here we describe the state of the art in the production of recombinant proteins in livestock with a focus on the mammary gland. The mammary gland is a promising site because of the ease of milk collection and the large amounts of protein that can be produced by specific gene promoters [ 5 , 7 ]. In 1989, John Clark of the Roslin Institute, Edinburgh (UK) demonstrated that the BLG promoter could be used to direct expression of the human coagulation factor IX gene in sheep and that the product was secreted during lactation [4].
For example, transgenic goats and cattle have been produced that secrete increased amounts of the antimicrobial protein lysozyme in their milk. Uroplakins are membrane-bound proteins specifically expressed in the differentiated uroepithelium of the bladder and urethra. While this may be suitable for some high-value proteins, the practical utility of the system remains to be demonstrated.
The protein processing capacity of the producing tissue, the persistence of foreign proteins in the sperm, and the ease of product purification are important considerations when choosing the site of production. Usually, these transgenes are fusions of the target protein gene with mammary gland-specific regulatory sequences (Figure 5.2.
Promotor Structural gene
Ideally, the transgenes should integrate stably into the host genome, be inherited in a Mendelian fashion, and direct the abundant expression and secretion of a desired protein to the target organ without affecting the health or well-being of the producing animal. The first requirement is a region of cloned DNA that codes for the amino acid sequence to be expressed (the structural gene. It may be necessary to change the coding sequence to optimize expression in a transgenic animal.
For example, if the protein is from an evolutionarily distant species, it may be necessary to change codons to match those most commonly used in the host. If the protein is not normally secreted, a signal peptide can be added at the N-terminus to direct secretion from the producing cell. Transgenes based on genomic sequences are usually expressed more consistently and abundantly than those based on cDNA.
Despite considerable knowledge of gene regulation, there is no standardized method for combining different genetic elements that can guarantee successful transgene expression. An important factor in the success of transgene expression is its location within the host genome.
Protein
The production of a biopharmaceutical in a transgenic animal requires that transcription and protein expression of the transgene be directed to the site of production. However, it is not uncommon for microinjection-produced founders to be mosaic-like due to the presence of the transgene. Mosaic components of a founder could potentially contain independent and distinct integrations of the transgene that segregate in the first generation.
Second, unlike oncoretroviruses, they do not require degradation of the nuclear envelope to gain access to the host genome. For more than two decades, ES cells have been powerful tools for experimental manipulation of the mammalian genome. Specific cyclin-dependent kinases, such as roscovitine, have been reported to increase the efficiency of the cloning process, although definitive evidence in terms of healthy offspring is lacking.
The purity of the protein preparation is an important factor in the evaluation of any transgenic product. At all times, documentation is essential and meticulous records must be kept of all activities, from the production of the DNA construct to the final product.
Translation of New Technologies in Biomedicines: Shaping the Road from Basic Research to Drug Development and Clinical
The selective, coordinated and intelligent use of the opportunities now being created will therefore be crucial. 14] discussed the importance of the interface between in chemico and in silico approaches in toxicology. An example of this is the use of the Meta Drug approach by Ekins et al.
The concept of the applicability domain, originally stated for in silico models, should also be applied to in vitro (and in vivo) tests. Recently, Casciano [59] reviewed the use of the omics technologies in model in vitro systems, with particular relevance to chemical-induced liver injury. The differences between rapidly perfused tissues (liver, kidney, brain) and slowly perfused tissues (muscles, skin) can be taken into account.
PBPK models can be used to guide dose selection, as well as identify responses to look for at later stages of the drug development process. This takes into account its structure and parameter values, using data not involved in the development of the model. 2007) Drug withdrawals in the United States: a systematic review of the evidence and analysis of trends.
2009 ) Use of the Integrated Discrete Multiple Organ Co-culture (IdMMOC ® ) system to assess multi-organ toxicity.
Part Two
Overview and Classifi cation of Approved Recombinant Drugs
Interestingly, it thus seems not possible to predict superiority for the more "authentic" or "natural" version of the glycoprotein. None of the established expression systems have general advantages in all situations and for all applications, and the choice of an expression system must be made on a case-by-case basis, sometimes with rather unexpected results. This in turn generates a high albumin affinity of the modified insulin from which it is slowly released.
Abatacept, a fusion protein consisting of the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to a modified Fc. Rilonacept, a dimeric fusion protein consisting of the ligand-binding domains of the extracellular portions of the human type I interleukin-1 receptor (IL-1RI) and IL-1 receptor accessory protein (IL-1RAcP) that is connected in line with the Fc. portion of a human IgG1. Alefacept, a dimeric fusion protein consisting of the extracellular domain of the human leukocyte function antigen-3 (LFA-3) linked in line with the Fc portion of human IgG1.
Follitropin is a heterodimeric protein composed of the glycohormone α-chain, which is also present in luteinizing hormone (LH), human choriogonadotropic hormone (hCG), thyrotropic hormone (TSH) and an FSH-specific β-chain. The fact is that it is extremely risky to develop such drugs, mainly due to the uncertainty associated with the curability of a particular disease with a biotech drug.
Downstream Processing
