Brief introduction to Biotechnology with reference to Pharmaceutical Sciences

1) Definitions of Biotechnology: Some of the definitions of biotechnology are mentioned below:

1. As per the Spinks Report (1980) biotechnology is defined as ‘the application of biological organisms, systems or processes to the manufacturing and service industries¹.
2. As per the United States, Congress’s Office of Technology Assessment biotechnology is ‘any technique that used living organisms to make or modify a product, to improve plants or animals or to develop microorganisms for specific uses¹.
3. Biotechnology means any scientific application that uses biological systems, living organisms, or derivatives thereof, to produce or alter products or processes for particular use’²⁵.

2) History of Biotechnology (in relation to Pharma industry)

The history of biotechnology can be divided into 3 phases:

1. Phase I (1980 to 1989): The major transformation of biotech started in the 1980s. During this period, recombinant proteins were used to treat major life-threatening diseases such as diabetes, anemia, etc. Also, during this period the hepatitis antigen vaccine was developed using recombinant DNA technology. During this period for the approval of a biotech product, the government followed the regulatory guidelines followed for a small drug molecule’s approval. However, the FDA officially fixed a separate guideline, application, and review process for the approval of biological/ biotech products. The biotech products developed during the 1980s were developed for endocrine and blood disorders. Insulin was the first product obtained by recombinant DNA technology which was approved by FDA⁷.
2. Phase II (1990 to 1999): During this period the pathophysiology of various diseases was clearly understood and the therapeutic potential of monoclonal antibodies was revealed. Due to advancements in molecular engineering, monoclonal antibodies (chimeric, humanized, and fully-humanized) were produced. Manipulation of the protein structure to obtain a protein which has therapeutically improved properties was also possible with molecular engineering. For example, Genentech’s thrombolytic alteplase used in the treatment of myocardial infarction was created by modifying the amino acid sequence and glycosylation of Tenecteplase. Due to this Tenecteplase could be administered by bolus instead of infusion. To accelerate the approval of biological products the regulatory authorities established the Prescription Drug User Fee Act (PDUFA) of 1992. In 1997 the Food and Drug Administration Modernization Act was brought in to enforce. Due to this act and PDUFA 1992, the approval process for biotech products was accelerated^7,8.
3. Phase III (2000 onwards): From 2000 onwards the evolution of the biotech industry started. Pegylation of protein molecules, improving pharmacokinetics and dosing schema were developed. For example, Pegylated compounds included interferon (peg-interferon alpha) for hepatitis C. Fusion protein technology became a major product development strategy. Full proteins (both recombinant and synthetic) were replaced by peptide molecules for diseases like diabetes and hereditary angioedema. Monoclonal antibody conjugates were produced due to which antibody served as a drug and delivery vehicle. Several orphan drugs were developed to treat genetic disorders and fatal diseases. During this period, the regulatory authorities focused on monitoring the adverse drug reactions, efficacy data, and the FDA established a new risk management plan. In the 2000s the major pharma companies either partnered or acquired the pharma companies⁷.

3) Scope and development of biotechnology with relation to pharma:

Most of the conventional medicines used for the treatment of various diseases were synthetic small size molecules. However, from the 1980s onwards the biotech startups-initiated research on biological molecules to check their therapeutic efficacy against diseases. Biotechnology is a multidisciplinary field of science and technology and its scope is extended to various sectors of science and technology. Biotechnology helps the pharma industry to develop new, safe, efficacious products. Since the 2000s application of biotechnology in the pharma industry is constantly increasing⁷.

4) Applications of Biotechnology in the pharma industry:

Some of the common applications of biotechnology in the pharma industry are discussed below:

1. DNA fingerprinting: The technique of DNA fingerprinting was discovered by Great Britain geneticist Alec J. Jeffrey in the year 1984. DNA fingerprinting is a technique used for authentication of various plant parts and herbal drug standardization. In DNA fingerprinting, bar-code like DNA patterns are generated after amplification of chromosomal DNA of the plant material. The sequence of DNA patterns produced is identified by DNA markers which are very specific for each plant species. Thus, DNA fingerprinting is an effective analytical tool used for herbal drug standardization, authentication of plant parts. In addition to herbal drug standardization DNA fingerprinting is also used to detect diseases cystic fibrosis, Huntington’s disease, hemophilia, etc. Detection of such diseases in the early stages increases the chances of patients been cured completely².

The various techniques used for DNA fingerprinting are listed below:

1. Hybridization based:
2. Variable Number Tandem Repeat (VNTR)
3. Probe hybridization with Micro and Minisatellite
4. Random Genomic Clone
5. cDNA Clone
6. Restriction Fragment Length Polymorphism (RFLP)
7. PCR based:
8. Inter Simple Sequence Repeat (ISSR)
9. Random Amplification Polymorphic DNA (RAPD)/Arbitrary Primed PCR
10. Amplified Fragment Length Polymorphism (AFLP)
11. DNA Amplification Fingerprinting (DAF)
12. Sequence-based
13. Simple Sequence Repeats (SSR)
14. Sequence Characterized Amplified Region (SCAR)
15. Cleaved Amplified Polymorphic Sequence (CAPS)
16. Single Nucleotide Polymorphism (SNP)².
17. Stem Cell in research and stem cell therapy:

Stem cells are undifferentiated cells that undergo division to give rise to a daughter cell which is similar to itself. These daughter cells further undergo division to form specific cells such as adipocytes, neuronal cells, macrophages, etc.

Diseases such as cancer and birth defects mainly arise because of abnormal cell division. Stem cells help in identifying the signals and mechanism of abnormal cell division and thus helps in proposing the new strategies to combat such diseases. Stem cells are used by researchers to screen for new potential drug molecules. For example, the cancer cell line is used to screen novel anti-cancer agents, macrophage cell line infected with Mycobacterium tuberculosis is used to screen potential antimycobacterial agents²⁵.

• Gene therapy: Gene therapy is one of the promising therapeutic techniques used in the treatment of cancer and other genetic disorders. Gene therapy is a technique to correct the abnormal genes. In gene therapy, a functional/ therapeutic is been transferred inside the target cells³. Vectors such as adenoviruses, simplex virus, liposomes, polyethylene glycol (PEG), gene gun are used for transferring the new gene inside the target cell. The new gene will ameliorate the abnormal metabolic event or the new gene will make a new functional protien^3,5.
• Transgenic plants: Transgenic plants is a class of genetically modified plants. The recombinant DNA also is known as rDNA technology which is used to produce medicinal plants with required traits and characteristics. Some of the significant traits possessed by transgenic plants are listed below:
• Higher production of secondary metabolites
• Production of plants resistant to pests, viruses, bacteria
• Drought resistant crop
• Production of hybrid seeds.
• Production of plants with an increased rate of photosynthesis
• Stress-resistant medicinal plants.

Example: Bacillus thuringiensis cotton also abbreviated as BT cotton is a genetically modified plant which is resistant to pest and insects. Bt cotton was produced by genetically modifying the cotton genome to express a microbial protein from Bacillus thuringiensis bacteria¹⁹.

• Diagnosis of diseases: With the help of various techniques in biotechnology, infectious and inherited diseases are detected. Some of the most commonly used detection techniques for the diagnosis of diseases are mentioned below:
• Agarose Gel Electrophoresis and Polyacrylamide gel electrophoresis
• Blotting techniques:
• Nucleic acid blotting
• Southern blot analysis
• Protein blotting
• Northern blot analysis
• Dot blot technique
• Autoradiography
• PCR-based techniques:
• Inter Simple Sequence Repeat (ISSR)
• Random Amplification Polymorphic DNA (RAPD)/Arbitrary Primed PCR
• Amplified Fragment Length Polymorphism (AFLP)
• Hybridization techniques
• Variable Number Tandem Repeat (VNTR)
• Probe hybridization with Micro and Minisatellite
• Restriction Fragment Length Polymorphism (RFLP)²⁵.
• Preparation of vaccines: Vaccines are biological preparations effective against a particular infectious disease. Vaccines work by providing active acquired immunity against the pathogen. The current advancement in biotechnology techniques, rDNA technologies, monoclonal antibodies techniques has brought strategies for vaccine development against infectious diseases. Eg: French microbiologists Albert Calmette and Camille Guerin in the year 1921 developed BCG (Bacillus Calmette-Guerin) vaccine against tuberculosis. In India, the first BCG vaccination drive was carried out in the year 1948⁴.
• Production of other biopharmaceuticals: Microorganisms such as bacteria, algae, viruses, fungi, yeast are one of the major sources for the production of biopharmaceuticals. Secondary metabolites obtained from microorganisms are reported to have significant therapeutic activity. With the development and advancement in biotechnology, rDNA technology these microorganisms can be easily manipulated to increase the production of the secondary metabolites. Biopharmaceuticals obtained from microorganisms are used as pharmaceutical excipients or as a therapeutic drug⁶. Few examples of the biopharmaceuticals obtained from microorganisms are listed in table 1.

Table 1: Examples of biopharmaceuticals used in pharma industry⁶  

Biopharmaceuticals	Source	Pharmaceutical use
Xanthan gum (polysaccharide)	Xanthomonas campestris	Used as a binder, matrix former, drug release modifier, viscosity enhancer, stabilizer, emulsifier, suspending agent, disintegrator, solubilizer, gelling agent, and bioadhesive.
Lutein	Green microalgae	Antioxidant, anti-inflammatory, and hepatoprotective effects.
Phycocyanin	Spirulina (Cyanobacteria)	Cosmetics, immunofluorescent techniques, antibody label.
Pyropheophytin a fucoxanthin	Brown macroalgae	Hepatoprotective

4) Conclusion:

• Biotechnology is a fusion of biological sciences and technology to produce products that are beneficial for human use.
• After 2000 biotechnology has made advancement in the field of rDNA technology, PCR, DNA fingerprinting, etc.
• Production of transgenic plants, growth hormones, and vaccines, stem cell therapy, gene therapy are some of the significant applications of biotechnology in the pharma industry.
• At present biotech products is one of the major sectors in the pharma industry.

5) Notations:

• Recombinant DNA technology: also known as rDNA technology is the process of joining two separate DNA molecules with help of DNA ligase. This rDNA molecule is inserted into the target cell²⁹.
• DNA ligase: is a class of enzyme that facilitates the attachment of DNA strands²⁸.
• Prescription Drug User Fee Act (PDUFA) 1992: was a law established by the United States Congress in the year 1992. This law authorizes the FDA to collect fees from companies that produce biological drug products. The main aim of this act was to expedite the drug approval process⁹.
• Agarose gel electrophoresis: Agarose gel electrophoresis is an analytical technique used to separate a mixture of macromolecules such as DNA, RNA, protiens¹⁰.  
• Nucleic acid blotting: Nucleic acid blotting is an analytical technique used to identify a specific gene or sequence of DNA or RNA¹³.  
• Southern blot analysis: is a hybridization technique used to locate a specific DNA sequence from the mixture of biological sample¹².
• Protein blotting: also known as Western blotting is an analytical technique used to identify a specific set of proteins in the biological sample¹¹.
• Northern blot analysis: is a hybridization technique used to locate a specific DNA sequence from the mixture of biological sample¹⁴.
• Dot blot technique: This technique is similar to other blotting techniques however, it doesn’t provide information regarding the size of the fragment. It is mainly used to identify a specific set of proteins in the biological sample¹⁵.
• Autoradiography: Autoradiography is a technique in which X-ray film is used to visualize the radioactively labeled fragments such as DNA¹⁶.
• Inter Simple Sequence Repeat: also abbreviated as ISSR is one of the PCR based techniques in which the DNA segments are amplified. ISSR is more dominant, stable, and repeatable²⁰.
• PCR: also known as polymerase chain reaction is a technique used to make multiple copies of sample DNA using DNA polymerase enzyme²².
• Random Amplification Polymorphic DNA: is based on the PCR principle which helps in to detect genetic variation¹⁷.
• Amplified Fragment Length Polymorphism: abbreviated as AFLP is a PCR based technique in which there is selective amplification of the subset of digested DNA fragments to produce and compare with fingerprints of interest genome²¹.
• Variable Number Tandem Repeat (VNTR): are short nucleotide sequences organized as tandem repeats at a specific location in DNA.
• Minisatellite: A minisatellite also known as VNTR is a repetitive sequence of 10 to 60 DNA base pairs which is repeated 5 to 50 times²³.
• Microsatellite: A microsatellite is a repetitive sequence of 1 to 6 DNA base pairs which are repeated 5 to 50 times²⁴.
• Restriction Fragment Length Polymorphism (RFLP): is a technique used in DNA fingerprinting. With the help of RFLP specific variations in the sequence of double-stranded DNA can be identified. Restriction endonucleases are the class of enzyme used in RFLP¹⁸.
• Restriction endonucleases are a class of enzyme that identifies a specific set of nucleotides (also known as restriction site) and cuts the DNA at that specific site¹⁸.
• Monoclonal antibodies: Monoclonal antibodies are man-made clones of parent immune cells. These proteins act as antibodies in the human immune system²⁶.
• Pegylation of proteins: is a biochemical technique used to modify proteins/ peptides, antibodies using polyethylene glycol (PEG)²⁷.

6) References:

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