Industrial Processes in Biotechnology PDF

1120-111 Biochemistry and Biotechnology Fundamentals Industrial processes 1120-111 Biochemistry and in biotechnology Biotechnology Fundamentals Industrial Processes...

1120-111 Biochemistry and Biotechnology Fundamentals Industrial processes 1120-111 Biochemistry and in biotechnology Biotechnology Fundamentals Industrial Processes in Biotechnology  1120-111 Contents Biochemistry and Biotechnology Definitions Fundamentals Industrial processes in biotechnology Production steps Factors affecting the selection of expression system Advantages and limitations of cultivation systems Contaminants from cultivation systems  1120-111 Biochemistry and Learning Objectives Biotechnology Fundamentals Industrial processes By the end of this lecture, You should be able to: in biotechnology Explain the process of recombinant protein production, including the major steps from gene isolation to protein extraction. Identify the key factors influencing the selection of an expression system for industrial biotechnology. Discuss the advantages and limitations of using different expression systems. Describe the phases of bacterial growth during fermentation and how they impact the production yield. Outline the methods used to identify and remove potential contaminants in biotechnological processes.  1120-111 Biochemistry and Biotechnology Biotechnology Fundamentals Industrial processes in biotechnology Biotechnology is a multidisciplinary field that involves the integration of natural sciences and engineering sciences to achieve the application of organisms for products and services (Genetic Engineering). It is the production process from raw materials with the aid of living organisms such as bacteria, and yeast, to perform specific tasks or produce valuable substances. Genetic engineering allows scientists to modify the genetic makeup of organisms to achieve desired outcomes. This can involve inserting genes from one organism into another, and consequently, create new traits or modifying existing ones. Used to produce vaccines, enzymes, insulin, antibodies.  1120-111 Biochemistry and Recombinant proteins Biotechnology Fundamentals Industrial processes in biotechnology They are proteins that are artificially produced using genetic engineering techniques. They have played a significant role in biomedical biotechnology, being used in research and as drugs in the treatment of various diseases. This is done through inserting a human gene into the genetic material of a common bacterium. This “recombinant” micro-organism could now produce the protein encoded by the human gene. The first recombinant protein used in treatment was recombinant human insulin in 1982. The recombinant protein industry has rapidly grown. To date, more than 130 recombinant proteins are approved by the US FDA for clinical use.  1120-111 Biochemistry and Recombinant proteins Biotechnology Fundamentals Industrial processes in biotechnology Recombinant proteins used in the clinic include recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs, and enzymes for treating major diseases such as diabetes, dwarfism, myocardial infarction, congestive heart failure, multiple sclerosis, neutropenia, thrombocytopenia, anemia, hepatitis, rheumatoid arthritis, asthma, Crohn’s disease, and cancers therapies.  1120-111 Plasmid Biochemistry and Biotechnology Fundamentals Industrial processes in biotechnology A plasmid is a small circular DNA molecule found in bacteria and some other microscopic organisms. Plasmids are physically separate from chromosomal DNA and replicate independently.  1120-111 Gene Biochemistry and Biotechnology Fundamentals A gene is a specific sequence of DNA that Industrial processes in biotechnology contains the necessary information to code for a functional product, usually a protein, but sometimes an RNA molecule (such as tRNA, rRNA, or regulatory RNAs). The lengths of the human protein-coding genes range from a few hundred bases up to a few millions. The shortest gene known is the tRNA (transfer RNA) gene, which typically consists of around 70 to 90 nucleotides The human insulin gene (INS) is a small gene of 1,425 base pairs.  1120-111 Biochemistry and Biotechnology Fundamentals Industrial processes in biotechnology Before the era of genetic engineering, most protein pharmaceuticals/hormones were obtained from animal sources (bovine or porcine). Potential drawbacks of proteins from animal sources: Do not always have same amino acid sequence as found in humans Inability to obtain large quantities of proteins from animal sources Inability to obtain products of high purity Immunological reactions are potential serious adverse effects  1120-111 Recombinant protein Biochemistry and Biotechnology production steps Fundamentals Industrial processes in biotechnology Step1- Isolating Gene and Vector (Bacterium Plasmid). Step2- Combining Gene and Vector Step3- Integrating Recombinant Vector into Host Cell Step4- Grow in culture medium Step5- Extract the protein  1120-111 Large-scale production of (biotechnology products) Biochemistry and Biotechnology used as pharmaceuticals. Fundamentals Industrial processes in biotechnology - Expression systems - Cultivation systems - Medium of cultivation - Removal of Potential contaminants - Downstream processing: (purification and recovery of biological products)  1120-111 Expression systems Biochemistry and Biotechnology Which Organism to Use?? Fundamentals Industrial processes in biotechnology Among microorganisms, host systems that are available include - Bacteria (Prokaryotic cells) - Yeast (Eukaryotic cells) - filamentous fungi - Mammalian cells - unicellular algae.  1120-111 The advantages of using E. coli as the Biochemistry and Biotechnology host organism Fundamentals Industrial processes in biotechnology 1) It has a fast growth kinetics. In glucose-salts media and given the optimal environmental conditions, its doubling time is about 20 min. 2) Rich complex media can be made from readily available and inexpensive components. 3) Plasmid transformation of E. coli can be performed in as little as 5 min  Limitation of mammalian expression 1120-111 Biochemistry and system Biotechnology Fundamentals 1) The system is highly expensive. Industrial processes in biotechnology 2) The complicated technology. 3) Potential contamination with animal viruses. But has many advantages 1) Post-Translational Modifications: Mammalian cells can glycosylate, phosphorylate, fold, and assemble proteins correctly, mimicking natural human processes. 2) High Biological Relevance: Proteins produced in mammalian cells are more likely to be bioactive and functional in humans. 3) Scalability: Suitable for both small-scale research and large-scale industrial production. 4) Common Host Cell Lines: Chinese Hamster Ovary (CHO) cells: Most widely used for therapeutic protein production. Today, production of biologics in mammalian cells dominates. Of the 58 biopharmaceutical products approved between 2006 and 2010, 32 were produced in mammalian cells, 17 were produced in E. coli, four in yeast, three in transgenic animals, and two in insect cultures.  1120-111 Cultivation systems Biochemistry and Biotechnology Fundamentals Industrial processes in biotechnology Large-scale manufacturing of therapeutic recombinant proteins is commonly performed in large capacity tanks (cultivation systems) referred to as * Fermenters or Bioreactors The bioreactor systems could be: 1) Laboratory < 50 L 2) Experimental 50 – 50,000 L 3) Industrial > 50,000 L  1120-111 Biochemistry and Biotechnology Fundamentals Industrial processes Three types of processes involving bioreactors in biotechnology The tank is filled with a The reactor is continually cultured cells remain in the starting material and cells in supplied with the starting reactor for 90 days and are medium, where incubation is material where equivalent supplied with fresh medium taking few hours to several amounts of the reaction and starting material on daily days for the biochemical mixture containing the end basis conversions. After which the product is taken out. tank is emptied, and the end products are purified.  1120-111 Components of the growth media Biochemistry and Biotechnology for mammalian cells Fundamentals Industrial processes in biotechnology Type of nutrient Example(s) Sugars glucose, lactose, sucrose, maltose, Fat fatty acids, triglycerides Water high quality sterile water Amino acids glutamine Electrolytes calcium, sodium, potassium, PO4- Vitamins ascorbic acid, -tocopherol, thiamine, riboflavin, folic acid Fetal calf Serum proteins: albumin, transferin Trace minerals iron, copper, cobalt, magnesium Hormones growth factors  1120-111 Cultivation systems Biochemistry and Biotechnology Fundamentals During the fermentation process, bacteria Industrial processes go through the following four phases in biotechnology 1) Lag phase: cells do not divide, but adapt to specific growth conditions in the medium 2) Exponential growth phase (Log Phase): active cell growth and genes are optimally expressed 3) Stationary phase: active cell growth slows down due to depletion of nutrients and spoilage of medium 4) Death phase: where bacteria die off  1120-111 Biochemistry and Biotechnology Primary metabolites Fundamentals Industrial processes in biotechnology Essential primary metabolites are synthesized by cells during growth phase, because they are essential for their growth (eg amino acids, vitamins). During cell growth, all that is required to produce primary metabolites is to optimize the growing conditions; as long as they proliferate, the amount of primary metabolites will increase. Secondary metabolites Secondary metabolites are only produced at late stage of the cell life as they are not needed for the actual growth process. Many of these secondary products are antibiotics and toxins. Secondary metabolites will peak after growth has stopped.  1120-111 Production yield of a recombinant Biochemistry and Biotechnology proteins can be optimized by Fundamentals Industrial processes in biotechnology 1- Reducing the time for the lag phase 2- Delaying the onset of stationary phase 3- Effective control of culture medium pH 4- Effective control of oxygen and temperature 5- Enforcing strict sterile measures and work protocols to prevent other microbial contamination.  1120-111 Potential contaminants arising from Biochemistry and Biotechnology cultivation process Fundamentals Industrial processes in biotechnology For pharmaceutical applications, purity of > 99 % is desirable 1. Viral contaminants can be removed by chromatography, inactivation by heat, or neutralization with specific antibodies. 2. Bacterial contaminants may elicit fever in humans and could be fatal can be removed by chromatography 3. Cellular DNA contaminants are DNA fragments which must be removed 4. Foreign protein contaminants: they are potential health hazards because they may be recognized as “antigens” by the patient receiving the recombinant protein; may lead to immune reaction.  1120-111 Criteria for ‘’Therapeutic proteins Biochemistry and Biotechnology produced by recombinant Protein Fundamentals technology’’ Industrial processes in biotechnology 1- Easily produced in large-scale and purified 2- Free of peptide and other potential contaminants 3- Have potentially fewer immune reactions 4- Meet ‘’Regulatory Standards’’ requirements  1120-111 Biochemistry and Biotechnology Fundamentals Industrial processes in biotechnology