The Application of Biotechnology - Essay Example

Overview of the Upstream and Downstream Processes for Biopharmaceuticals Real Time Quality Control Overview of the Upstream and Downstream Processes for Biopharmaceuticals Real Time Quality Control The application of biotechnology has opened new realms that have presented opportunities for the large-scale production of drugs. However, the production involves multiple processes that range from the upstream processing and to the downstream processing that ensures that the product undergoes purification to the required degree and eventually packaging. The focus of this essay will be on the upstream and downstream processing, and then discuss the significance of real time quality control and product analysis technology. In a broad definition, upstream processes refer to all the procedures that take place prior to the collection of host cells. However, a more specific approach of the upstream processes will be adopted in this essay. Downstream processes refer to the processes that take place after the fermentation process of purification to the packaging of the Biopharmaceuticals. Moreover, the issue of quality control techniques in a bid to ensure successful product analysis will receive attention in this essay. Upstream Processing One of the critical steps in the upstream processing is the choice of an effective culture and expression system.There are three types of culture systems that may be adopted in the fermentation process. One of them is the batch culture, which is the commonest culture system that used in many industries because of lack of complications. The fact that batch bioreactors are easily available explains why many companies have exhibited preference for it. Batch cultures need to be loaded with all the required nutrients and substrates, and then inoculated with the selected microorganism. Although it is commonly referred to a closed system, there is an evident need to maintain a measure of exerting control on factors such as pH and aeration. After fermentation runs to completion, product removal follows (Boudreau & McMillan, 2007). Although the batch system has been highly preferred, it presents certain challenges, especially because of the surging lack of constancy in the producing formation. In the initial phase when the microorganism is undergoing growth, there is no productivity. Moreover, accumulation of the product also limits further production (Buckel, 2001). In other cases, the presence of a high substrate concentration in the initial phase serves as a form of inhibition. In a bid to address this challenge, the development if fed batch where an inflow is introduced was a benchmark of success. Although the batch culture still has some of the outstanding disadvantages of the batch culture, the fed-batch presents an increased working period, ensuring a higher rate of productivity (Buono, 1995). Of critical importance is the determination of the appropriate rate of input of the nutrients, a factor that affects the rate of production.Experts have highlighted that the fed batch needs a salient understanding of the physiology of the strains utilized in the fermentation process, as well as a caution against any form of contamination that may result from the inflow if nutrients. In addition, the development of the continuous culture has been a hallmark of fermentation success. Although it requires a unique bioreactor that can handle both the inflow and outflow of nutrients, it presents certain desirable features as it ensures a constant rate of production. A continuous culture system opens up the possibility of automating the production of bio products, a factor that is highly desirable especially in the production of biodrugs. This type of culture system ensures that an evident and constant monitoring of the process is possible. With such monitoring, it is possible to undertake quality control measures(Rehbinder, 2009). However, maintain a steady state requires expertise, and measures if minimizing the rate of contamination are needed. Maintaining sterility may prove difficult in continuous cultures, posing challenges in the management of quality in the production of Biopharmaceuticals, an aspect that should not receive any form of compromise in the manufacture of biodrugs. After determination of the appropriate culture system, an effective expression system is critical. Research has helped in the analysis of different expression systems, although the choice depends on the pharmaceutical of interest. Bacterial cells have been a preferred expression system in many cases because of the salient understanding of their manipulation. With an immense wealth of knowledge on the DNA sequences of bacterial DNA, plasmid vectors suitable for transferring the genes into the bacterial DNA. Moreover, with advanced understanding of the culture conditions, it is evident that bacterial cells can be cultured easily in large quantities. However, bacterial cells pose certain disadvantages when used as expression systems. They lack the potential to produce complex proteins, and even if they do, there are limitations in the posttranslational modifications. More concerns surround the inability of protein to fold, leading to the formation of inactive proteins (Newton, 2007). In the case of heterologous Biopharmaceuticals, it is challenging to rely on bacterial cells as expression systems. On the other hand, yeast cells firm the second category of expression systems used in the large-scale production of recombinant drugs. The fact that there is an advanced level of understanding their physiology and genetic constitution makes it easier to manipulate them genetically through the introduction of foreign genes (Eib, & Eibl, 2013).  Moreover, there are multiple promoters that whose isolation have occurred successfully, in addition to the presence of a diverse range of plasmids, factors that increase the preference of yeast cells as expression systems (Stacey & Davis, 2007). Although yeast cells are eukaryotes and present an advantage over bacterial cells as they allow a level of p [post translational modification and folding of proteins to take place, this proves impossible when complex protein is involved. This only translates to the fact that the biodrugs produced using yeast cells may be inactive because of the lack of effect posttranslational modification (Doran, 2013). Insect cells have also been considered as a better expression system compared to yeast and bacterial cells. With insect cells, experts have highlighted that the significance of the baculovirus vector cannot receive any form of underestimation (“Recombinant Drug Development, Regulation, and Commercialization”, 2011). This virus exhibits a remarkable capacity to infect cells and undergo rigorous multiplication. In the process of its multiplication, it transfers the gene of interest into the insect cells (Kristiansen & Ratledge, 2007). Insect cells are the preferred expression systems in the production of multiple biodrugs because of the salient possibility of successful posttranslational modifications and protein folding. In the case where insect cells are used as the preferred expression system, the protein of interest is usually released into the medium or within the cell as a fusion protein (Turner, 2010). Mammalian cells from the last category of expression system in the manufacture of recombinant drugs. The capacity of mammalian cells to produce vertebrate proteins that have undergone complete posttranslational modification and folding presents the assurance of active recombinant drugs. Current research focuses on describing the most effective vectors and promoters, in a bid to increase the level of productivity, as the current level of productivity is minimal (Gellissen, 2004). An additional upstream process is the recombinant gene technology whereby the gene of interest must be sequenced, and insertedinto a selected vector. With the available advanced understanding of restriction enzymes, recombinant gene technology has proven highly viable. It is important for the right vector to be selected, and measures of ensuring that the recombinant vector DNA is incorporated into host cells. Prior to the beginning of the production process, transformed cells of the selected expression system must be present (Zanders, 2011). Downstream processing After the completion of the fermentation process, the biodrugs are isolated either from the medium of from the cell and purified. The level of purification depends on the level of purity required for the biodrug, without doubt, recombinant drugs are required in a high degree of purity. Therefore, a rigorous process of purification must be undertaken in order to achieve the desired purity. In many cases, an assay technique is used to determine whether the fermentation process has yielded the protein of interest, in this case the recombinant drug. In case the biodrugs are released into the cell, there is a salient need to undertake cell fractionation in a bid to separate different cell components. Include salting out, which makes use of different salt concentration in a bid to concentrate the biodrug (Kristiansen & Ratledge, 2007). In other cases, dialysis is preferred. Other critical techniques used at this phase. Notably, the fractionation system may be prone to error as it is a matter of trying. The following chart shows some of the steps: Homogenate formation→ disruption of the cell membrane → centrifugation → obtained supernatant → second round of centrifugation →differential centrifugation. Purification techniques Gel filtration is one of the purification techniques used in the purification of recombinant drugs (Hambleton, 2004). The basis of this technique is the separation of proteins according to their different sizes by applying the sample of the biodrugat the top of a beaded column. In addition, the potential of ion exchange chromatography in the case of charged recombinantproteins, ensuring their separation according to their pertinent charges (Dunford, 2012). Affinity chromatography that employs different chemical groups, and hence ensures separation of the protein according to their affinity levels to the groups. High-pressurechromatography is an additional technique used in protein purification. Other preferred techniques include isoelectric focusing and gel electrophoresis, which utilizes a gel column and separates protein according to size and charge. After the purification process, packaging of the biodrugs follows. Typical purification chart Fermentation → sonication →membrane fractionation → heat treatment →ion exchange chromatography →gel filtration. Steps in membrane fractionation Sonication → centrifugation (supernantant) →ultra centrifugation → precipitate membrane →wash membrane → dissolve membrane with buffer → ultra centrifugation → supernatant collection →membrane fractions. Overview of Real Time Quality Control in Process analytical Technology Quality control is one of the critical aspects in the production of biodrugs. The Food and Drug Administration (FDA) has a unique mandate in ensuring that all industries act in compliance with the defined standard ofpromoting quality. In essence, the FDA has to approve any recombinant drug before it proceeds to the market. However, there is a need for a real time quality control routine during the manufacture of biodrugs. This is because quality assessment after the completion of the fermentation process highlights the mistakes that are irreversible, and the entire batch products are disposedof (Troy, 2005). This places emphasis on the salient need to adopt a real time quality monitoring that ensures that any event that may affect quality is noted in good time, allowing time to make amends. For example, it is possible for midpoint quality checks reveal any occurrences that affect quality. However, despite the criticality of adopting real time quality control checks, there are multiple uncertainties surrounding the issue. Although there is evidence of successful application of the use of real time quality control in other pharmaceutical manufacturing firms, the application of such processes in the manufacture of biopharmaceutical presents certain challenges. Since Biopharmaceuticals are protein molecules, it may take time to analyse the proteins as protein biochemical tests take a longer period of time before they yield results. Such challenges explain the uncertainty that surrounds the application of realtime quality control despite its obvious necessity (Ho & Gibaldi, 2013). Currently, research focuses on defining ways of incorporating PAT techniques into the manufacture of recombinant drugs, as in the future, such techniques will be fundamental features in the manufacture of biophramaceuticals. Although many industries have expressed their fears in adopting the real time analytics, there is evidence of positive signs as revealed by the concerted efforts of different industries and researchers (OKeefe, 2000). Conclusion Evidently, the manufacture of biopharmaceuticals has only become a reality because of the advances made in understanding recombinant gene technology and the fermentation process. With the description of different culture systems, expression systems, and availability of arrange of bioreactors, it is possible for manufacturers to adopt a combination that presents the capacity to yield a large-scale production of biodrugs. The determination of such combinations defines the upstream processing. Downstream processing ensures that the purity of the produced biodrugs is of the required degree. Quality control is a critical aspect in the production of Biopharmaceuticals, and there is an emphasis on the real time quality control methods. 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New York: Springer. Zanders, E. D. (2011). The science and business of drug discovery: Demystifying the jargon. New York: Springer. Read More