Pharmaceutical Biotechnology and Recombinant DNA Technology

A biosensor is an analytical device containing immobilized biological material (enzymes, antibodies, nucleic acid, hormone) which specifically interact with an analyte and produce physical, chemical, or electrical signals that can be measured. An analyte is a compound (e.g., glucose, urea, drug, pesticide) whose concentration has to be measured.

Biosensors consist of two main components: a "sensing element" and "transducers."

• Sensing element: May be either enzymes, antibodies, DNA, tissues, or whole cells.
• Transducers: Convert a biochemical reaction or response into electrical signals.

The basic components of biosensors are: analyte (sample), sensing element, and transducer.

Principle of Biosensors

The analyte, on binding to the biological material, forms a bound analyte that produces a response. The analyte converts into a product, and changes associated with this product are transformed by the transducer into electrical signals that are amplified and measured.

Biosensor Types and Applications

The various types of biosensors are:

1. Electrochemical Biosensors
2. Thermometric Biosensors
3. Optical Biosensors
4. Piezoelectric Biosensors

Protein Engineering

Protein engineering is a biotechnological process that involves altering the sequence of amino acids in a protein to create new proteins or improve existing ones.

Protein engineering develops proteins having desired functions by manipulating their stability and specificity. It can be done by taking two main approaches.

The techniques used in protein engineering are classified into two basic categories:

1. Genetic modification
2. Chemical modification

1. Genetic Modifications

Modifications of protein through genes is an easy and more efficient approach. The basic method includes modifications of the responsible gene to generate the protein with novel properties.

• Synthetic oligonucleotides (small DNA/RNA fragments) are used for in vitro mutagenesis.
• A small oligonucleotide primer is synthesized with the desired modifications.
• Allows the hybridization of the oligonucleotide primer at the appropriate site of the parent gene.

2. Chemical Modification

Chemical modification is the most widely used method. In this method, the functional group on the side chain of a natural enzyme is changed, or parts of the original protein are modified and replaced.

Protein modification is used to increase the stability of enzymes to high temperatures and organic solvents.

Genetic Engineering

Genetic engineering or Recombinant DNA technology is the field of biotechnology that specializes in developing new combinations of genetic material, called recombinant DNA (rDNA), which are artificially constructed in the laboratory for introduction into host cells for propagation and multiplication.

The various tools required for rDNA or genetic engineering are:

• Restriction enzymes
• Source of donor DNA
• Reverse transcriptase and polymerase enzymes
• Vectors and host organisms

Applications of Genetic Engineering

1. Genetic engineering is used to create drugs like insulin, human growth hormones, and monoclonal antibodies.
2. Used in gene therapy to treat various diseases.
3. Model animals: Genetic engineering can create animals that mimic human conditions.
4. Genetic engineering can be used to create vaccines.
5. Genetic engineering can be used to create disease-resistant crops and improve the nutritional value of crops.
6. Genetic engineering can be used to improve the health of livestock.
7. Genetic engineering can be used in the study of defective genes at the fetus stage.

Amylase

Amylase is an enzyme that breaks down starch into sugar.

Amylase is present in human saliva. All amylases are glycoside hydrolases which act on α-1,4-glycosidic bonds.

They are specifically used in the industrial starch conversion process.

The α-amylase is obtained from plants, animals, or microbes. This enzyme shows its activity in a wide range of pH and temperature.

Enzyme Immobilization

Enzyme immobilization is a technique of restraining enzymes on an inert support for their stability and functional reuse.

The first commercial application of immobilized enzyme technology was realized in 1969 in Japan with the use of Aspergillus oryzae aminoacylase for the industrial production of L-amino acids.

Advantages of Enzyme Immobilization

• The immobilization process can lead to increased activity and stability of the enzyme molecules.
• Immobilized enzymes are easily recovered from the reaction mixture and reused.
• The enzyme immobilization process avoids contamination in products and increases the enzyme-to-substrate ratio.

Methods of Enzyme Immobilization

Enzymes can be immobilized by the following methods depending on the physical relationship of the catalyst being used with the carrier matrix:

1. Adsorption Method
2. Covalent Method
3. Entrapment Method
4. Encapsulation Method

1. Adsorption Method

Adsorption of an enzyme is brought about by allowing the enzyme to come in contact with a polymer support.

The enzyme molecules adhere to the surface of the carrier matrix with the help of hydrophobic bonds and by forming several salt-linkages per enzyme molecule.

Various forces like van der Waals and electrostatic interactions to some specific ligand attached to the carrier matrix are involved in the adsorption method.

2. Covalent Method

A covalent bond is formed between the chemical groups of the enzyme and the chemical groups on the surface of the carrier.

Covalent bonding has the advantage of an attachment not reversed by pH, ionic strength, or substrate.

3. Entrapment Method

Entrapment is a phenomenon in which the enzyme molecules are held or entrapped within appropriate fibers or gels. This entrapment, however, may or may not be the result of covalent bonding existing between the enzyme entities (molecules) and the carrier matrix.

In this method of enzyme immobilization, the enzyme molecules are either held or entrapped within fibers or gels. Example: Penicillin acylase is a fiber-entrapped enzyme that was immobilized by entrapment in the microcavities of synthetic fibers.

4. Encapsulation Method

Encapsulation is a versatile and effective method of enzyme immobilization by entrapping. In this method, the enzyme molecules are regularly taken up in an aqueous medium and confined in a semi-permeable membrane, which allows absolute free movement of the enzymes.

Pharmaceutical Biotechnology Scope

The various scopes and applications of pharmaceutical biotechnology are:

• Recombinant DNA technology
• Gene therapy
• Criminal forensics
• Monoclonal antibodies
• Genetically engineered vaccines
• Plant tissue culture
• Genetic testing

Genetic Testing

This involves direct examination of the DNA and is used for:

• Determining sex
• Forensic / identity testing
• Newborn screening
• Prenatal diagnostic screening
• Pre-symptomatic testing for determining the risk of developing cancers

What is Biotechnology?

Biotechnology is the branch of science that combines biology and technology with the aim of improving people's quality of life. It uses living cells or any of their components to develop products with specific aims.

Applications include:

• Recombinant DNA technology
• Gene therapy
• Criminal forensics
• Monoclonal antibodies
• Genetically engineered vaccines
• Plant tissue culture

Industrial Use of Amylase and Protease

Amylase: Amylase is an enzyme that breaks down starch into sugar.

Amylase is present in human saliva. All amylases are glycoside hydrolases which act on α-1,4-glycosidic bonds.

They are specifically used in the industrial starch conversion process.

The α-amylase is obtained from plants, animals, or microbes. This enzyme shows its activity in a wide range of pH and temperature.

Enzyme Immobilization: Enzyme immobilization is a technique of restraining enzymes on an inert support for their stability and functional reuse.

The first commercial application of immobilized enzyme technology was realized in 1969 in Japan with the use of Aspergillus oryzae aminoacylase for the industrial production of L-amino acids.

Genetic Engineering: Genetic engineering or Recombinant DNA technology is the field of biotechnology that specializes in developing new combinations of genetic material, called recombinant DNA (rDNA), which are artificially constructed in the laboratory for introduction into host cells for propagation and multiplication.

The various tools required for rDNA or genetic engineering are:

• Restriction enzymes
• Source of donor DNA
• Reverse transcriptase and polymerase enzymes
• Vectors and host organisms

Recombinant DNA Technology

Recombinant DNA technology or genetic engineering is the field of biotechnology that specializes in developing new combinations of genetic material, called recombinant DNA (rDNA), which are artificially constructed in the laboratory for introduction into host cells for propagation and multiplication.

Steps in Recombinant DNA Technology

1. Isolation of Genetic Material or DNA:
   • Location: Genetic material is present inside the cells. It has to be obtained in pure form without even the attached histones and other proteins.
   • Treatment: To get the desired DNA, cells are treated with lysozyme (for bacteria), cellulase and pectinase (for plant cells), and chitinase (for fungal cells).
   • Process: The treated cells are homogenized and centrifuged to rupture the cells as well as nuclear envelopes. The homogenized product is then treated with proteases (for digesting histones and other proteins) and ribonucleases (for digestion of RNAs).
   • Precipitation: DNA is precipitated by the addition of chilled ethyl alcohol. DNA appears as a mass of very fine threads.
2. Cutting of DNA at Specific Locations and Its Pasting:
   • Digestion: Purified DNA molecules are subjected to restriction enzymes. The progress of restriction enzyme digestion is checked through agarose gel electrophoresis.
   • Passenger DNA: The genetic material which is meant for transfer to another organism is called passenger DNA.
3. Amplification of Gene:
   • Method: Amplification of the gene of interest is done by PCR (Polymerase Chain Reaction).
   • Definition: PCR is the in vitro synthesis of multiple copies of a gene or DNA segment.
4. Insertion of Recombinant DNA into Host Cell/Organism:
   • Methods: Both direct and indirect methods are known to introduce the desired genes into host cells.
   • Common Process: In a common method, the desired gene is ligated to a plasmid vector and introduced into bacteria (E. coli).

PCR and Its Applications

PCR or Polymerase Chain Reaction is the in vitro synthesis of multiple copies of a gene or DNA segment.

It uses two sets of small chemically synthesized oligonucleotide primers and a thermostable DNA polymerase like Taq Polymerase.

There are 3 basic steps in PCR:

1. Denaturation: The target DNA is heated to about 94°C. It separates the two strands which are now ready to act as templates.
2. Annealing: The oligonucleotide primers anneal or hybridize to each of the single-stranded DNA templates at its 3' end. This step is performed at a lower temperature.
3. Extension: The thermostable DNA polymerase extends the primers using dNTPs.

Applications of PCR

• For DNA amplification
• Detecting mutations
• Diagnosing genetic disorders
• Producing in vitro mutations
• For preparing DNA for sequencing
• Analyzing genetic defects in single cells from human embryos
• Identifying viruses and bacteria in infectious diseases
• For characterization of genotypes

Recombinant Insulin Production

Human insulin was the first recombinant-derived product used for the treatment of diabetes.

Two DNA sequences were prepared by Eli Lilly for the two chains, A (for 21 amino acids) and B (for 30 amino acids) of insulin by reverse transcription of their mRNAs.

Plasmids of E. coli and the insulin gene are treated with the same restriction endonuclease. The two are joined together by DNA ligase.

It produces rDNA in the form of plasmids carrying the insulin genes.

Insulin genes are attached in the vector plasmids adjacent to the lacZ gene encoding β-galactosidase.

A culture of plasmid-free E. coli is now inoculated with the recombinant plasmid.

The genetically engineered bacteria are tested for the formation of a fusion polypeptide consisting of one insulin subunit and a β-galactosidase sequence.

As the presence of both insulin subunits is ensured, the bacteria are first multiplied and then introduced into a sterilized bioreactor containing the growth medium.

The latter are then mixed. Insulin is formed. Since it is exactly similar to human insulin, it is also called humulin.

Principle of PCR

PCR is the Polymerase Chain Reaction. It is the in vitro synthesis of multiple copies of a gene or DNA segment.

The basic principle of PCR is to amplify a DNA segment by repeating cycles of heating and cooling.

Processing and Storage of Blood and Plasma

Whole Human Blood: This is the simplest and most common type of blood donation. The whole blood is transfused for replacing the red blood cells, clotting factors, or other normal constituents missing from the patient's blood.

Processing: The processing of whole human blood includes:

Blood is drawn from a donor and stored in a monitored refrigerator at 1-6°C temperature for 21 days (if collected in CPD (citrate phosphate dextrose anticoagulant solution)) or for 35 days if collected in CPDA-1 (citrate phosphate dextrose adenine solution).

Storage: The storage requirements for whole human blood are:

• Soon after the blood is collected, it should be cooled to 4-6°C temperature and maintained at that temperature for 21 days.
• The concentration of hemoglobin in the blood-anticoagulant mixture should be not less than 9.7 g/dL.
• The blood cells and the serum should be examined to determine the ABO blood group, and the cells alone should be examined for the Rh group.
• The blood sterility should be checked before it is used.
• The label should bear the ABO and Rh groups, volume of blood and anticoagulant solution present, date of collecting blood, and required storage conditions.

Dried Plasma: This has several advantages as compared to whole blood:

• It can be given to patients of any blood group.
• It is well stored for 5 years.
• It could be stored at room temperature (below 20°C) if protected from light.

Processing: Dried plasma is prepared from time-expired citrated blood. The supernatant fluid is separated by centrifugation. Batches of not more than 10 bottles are pooled and are kept at 4 to 6°C while samples are tested for sterility. The pools which have passed the test for sterility are then redistributed in transfusion bottles.

Storage:

• It should be stored under dry conditions and away from light at 25°C.
• The name and percentage of anticoagulant and other added substances should be mentioned.
• The content should not be used after 3 hours of reconstitution.

Large-Scale Fermenter Design

A fermenter is a system consisting of different types of equipment to provide microbial growth by controlling environmental conditions.

The various design aspects required to be considered in constructing an ideal fermenter for the production of pharmaceuticals are:

• Provide operations free from contamination.
• Adequate mixing and aeration.
• Maintain specific temperature and pH.
• Minimize liquid loss from the fermenter.
• Monitoring and control of dissolved oxygen.

Fermentation Methods and Requirements

The process of fermentation involves the biochemical activity of organisms during their growth, development, reproduction, and death. Fermentation technology employs organisms to produce food, pharmaceuticals, and alcoholic beverages in industries on a large scale.

The 3 different processes or methods of fermentation are:

• Batch fermentation: In the batch fermentation process, the microorganisms are inoculated in a fixed volume of batch culture medium. The organisms during their growth consume the nutrients, and growth products start accumulating. It consists of 4 phases: lag phase, transient phase, exponential phase, and deceleration phase. In batch fermentation, exponential growth occurs for a limited period; with the change in nutrient conditions, the growth rate decreases and begins the deceleration phase.
• Fed-batch fermentation: In the fed-batch fermentation process, freshly prepared culture media is added periodically without removing the culture fluid. This type of fermentation is used for producing proteins from recombinant microorganisms.
• Continuous fermentation: In this, near-balanced growth is obtained with little fluctuation in nutrients, metabolites, or biomass. In this process, fresh medium is added to the batch process at the exponential phase of growth, and the medium is withdrawn along with cells. Continuous methods of cultivation allow the organisms to grow under steady-state conditions, in which growth occurs at a constant rate in a constant environment.

General Requirements: The general requirements for microorganism growth or fermentation are essential for maintaining optimal yield and productivity.

Vitamin B12 Fermentation Production

Vitamin B₁₂ is produced using many different microbes. On a commercial or large scale, Pseudomonas denitrificans is used.

Fermentation of P. denitrificans uses sugar beet molasses containing 5-10% betaine (trimethyl glycine) to serve as the carbohydrate source. Betaine stimulates vitamin B₁₂ production. The growth of Pseudomonas and vitamin B₁₂ biosynthesis are carried out under aerated conditions with agitation.

Lab-scale fermentation of vitamin B₁₂ using P. denitrificans occurs via:

1. Maintenance culture: Incubated for 4 days at 28°C.
2. Seed culture: Incubated for 3 days at 28°C without agar.
3. Production culture: Incubated with seed culture for 90 hours at 29°C.

Isolation and Purification: Cells are separated from the culture broth and lysed by heating at 80-120°C for 10-30 minutes. The pH should be maintained at 6.5-8.5. The lysed cells release various cobalamins, which are solubilized with potassium cyanide in the presence of sodium nitrite. All the cobalamins are converted to cyanocobalamin (Vitamin B₁₂).

Storage of Whole Human Blood:

• As soon as blood is collected, it should be cooled to 4-6°C temperature and maintained at that temperature for 21 days.
• The concentration of hemoglobin in the blood-anticoagulant mixture should be not less than 9.7 g/dL.
• The blood sterility should be checked before it is used.

Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-Linked Immunosorbent Assay (ELISA) is one of the most essential immuno-enzyme assays. In this method, the antigen is attached to an antibody anchored on a solid phase. This helps in retaining the immunological as well as enzymatic activity. ELISA is also referred to as a qualitative or quantitative assay for antibodies.

Mutations and Mutagens

Mutation is defined as "an abrupt alteration in the phenotype of an individual." Examples of mutations include point mutations, frameshift mutations, etc.

Mutagens (agents of mutation) are those physical or chemical agents that increase the frequency of mutations. Examples include X-rays, γ-rays, alkylating agents, etc.

Types of Mutations

1. Frameshift Mutation: In this type, addition or removal of DNA bases alters a reading frame of genes that comprises groups of 3 bases, each coding for a single amino acid. This mutation shifts the grouping of these bases and alters the amino acids coding to form a non-functional protein. Frameshift mutations include insertions, deletions, and duplications.
2. Inversion: Occasionally, a region of DNA reversibly (but still inherited) aligns into the opposite orientation. For example, variations in the flagellar antigens are seen in Salmonella typhimurium.
3. Substitution Mutation: In this type, an exchange between two bases occurs. This alters a codon encoding a different amino acid, thus producing a different protein. For example, in sickle cell anemia, substitution occurs in a codon (GAG mutates to GUG and changes Glu to Val).

Western Blotting

The method of Western blot was discovered by George Stark at Stanford. Western blotting, also known as immunoblot or protein blot, detects specific proteins in a sample of tissue extract.

Western blotting is a fast and sensitive method of assay that detects and characterizes proteins. This technique requires SDS-PAGE.

Principle of Western Blotting

It works by the immunochromatography principle, wherein separation of proteins as per their molecular weight occurs in polyacrylamide gel. The separated proteins are transferred onto a nitrocellulose membrane and then detected by a specific primary antibody and a secondary enzyme-labeled antibody and substrate.

Southern Blotting

E.M. Southern invented the technique of Southern blotting. This method is useful in molecular biology. In this method, hybridization analysis is performed by transferring the DNA from a gel to a membrane.

Role of Transduction in Pharmacy

Transduction is a genetic recombination method which involves the transfer of genetic material from a donor to a recipient cell through a non-replicating bacteriophage. This process was discovered by Joshua Lederberg and Norton Zinder in 1952.

The transduction process is of two types:

1. Generalized transduction: In this, a DNA fragment is transferred from one bacterium to another by a lytic bacteriophage (virulent) which carries donor bacterial DNA due to an error.
2. Specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage which carries donor DNA along with the phage genome due to an error. In this, the genome is inserted at a specific site.

Applications of Transduction in Pharmacy

1. Gene Therapy and Viral Vectors: Transduction is a fundamental process in gene therapy, where viral vectors are used to deliver therapeutic genes into a patient's cells. This allows the correction of genetic defects or the introduction of genes that can produce therapeutic proteins.
2. Vaccine Development: Some viral vaccines utilize transduction principles, where a modified virus is used to deliver viral antigens into the host cells, stimulating an immune response without causing disease.
3. Drug Delivery and Targeted Therapy: Transduction can be used to develop targeted drug delivery systems, where viral vectors are modified to deliver therapeutic drugs or genes to specific tissues or cells, minimizing off-target effects and improving drug efficacy.
4. Genetic Engineering: Transduction is a powerful tool in genetic engineering, allowing for the modification of genes in bacteria and other microorganisms, facilitating the production of pharmaceuticals.
5. Somatic Cell Genetics: Transduction plays a role in somatic cell genetics, where it can be used to modify the genes of cells in the body, potentially for therapeutic applications.

Microbial Biotransformation

Microbial transformation is a biological process in which organic compounds are modified into reversible products.

These biotransformation reactions are catalyzed by purified enzymes present in microbial cells or pure cultures of microorganisms.

Biotransformation is the process in which "microorganisms convert organic compounds into structurally related products."

It can also be said that biotransformation involves microbial conversion of a substrate into a product with a few enzymatic reactions.

Types of Biotransformation Reactions

1. Oxidation: If a secondary hydroxyl group is located between a primary and secondary group, which is in the cis-position with respect to the oxidizable group, then the hydroxyl group is oxidized to give a ketone. For example, conversion of naphthalene into salicylic acid by oxidation by Pseudomonas bacterium.
2. Reduction: Hydroxyl groups at C6, C11, and C17, an α-methyl group at C16, and unsaturations lead to a decrease in the reduction rate in steroids. The presence of electron-withdrawing groups at C in a 3-keto substrate shifts the direction from oxidation to reduction. Reduction of C2 ketone occurs in the presence of Streptomyces bacterium.
3. Hydrolysis: A large number of esters, glycosides, epoxides, lactones, β-lactams, and amides can be hydrolyzed by microorganisms. For example, tartaric acid is produced from maleic anhydride by Achromobacter tartarogenes.
   Maleic anhydride → tartaric acid
4. Condensation: Microbial condensation was utilized in 1934 in the synthesis of natural ephedrine. Acetaldehyde reacts with benzaldehyde in the presence of fermenting yeast and gives (R)-1-phenyl-1-hydroxy-2-propanone. The propanone undergoes reductive condensation with methylamine to yield (1R, 2S)-ephedrine.

Plasmids as Cloning Vectors

Apart from the bacterial chromosome (nucleoid), genetic elements are also found to be present in the cytoplasm of bacterial cells. These genetic elements are called plasmids.

Many bacteria contain additional plasmids that range in size from several to 100 kbp.

Most plasmids are circular, double-stranded DNA molecules varying from 1,500 to 400,000 base pairs.

Transposons are genetic elements that contain several kbp of DNA, including the information necessary for their migration from one genetic locus to another.

Citric Acid Fermentation Production

Microorganisms: Mostly produced using the filamentous fungus Aspergillus niger or Aspergillus clavatus.

Raw Materials / Medium Composition:

• Carbohydrate Source: Beet molasses, cane molasses, sucrose, glucose, or starch hydrolysates. Molasses is preferred due to its low cost and high sugar content.
• Nitrogen Source: Ammonium nitrate, ammonium sulfate, or urea to encourage growth and acid production.
• Inorganic Salts & Minerals: Potassium dihydrogen phosphate (KH₂PO₄) and magnesium sulfate (MgSO₄).

Fermentation Methods:

1. Surface Culture Method: The medium is placed in shallow pans, and the fungus grows as a mat on the surface.
2. Submerged Culture Method: This is the primary industrial method. Fermentation is carried out in large-scale stirred tank bioreactors.

Fermentation Conditions (Submerged):

• Aeration & Agitation: Continuous and vigorous aeration is required since Aspergillus niger is strictly aerobic.
• Temperature: Maintained precisely between 25-30°C.
• pH: Maintained at a very low range of 2.0-3.0. A low pH is critical because it minimizes the risk of bacterial contamination and suppresses the unwanted formation of oxalic acid and gluconic acid.
• Duration: Typically runs for 5-8 days.

Large-Scale Penicillin Production

Introduction: Penicillin is a widely used β-lactam antibiotic. Originally discovered by Alexander Fleming in 1928 from Penicillium notatum, commercial large-scale production utilizes high-yielding mutant strains of Penicillium chrysogenum.

Inoculum Development: Spores from a pure master stock culture are germinated on agar slants, transferred to shake flasks, and sequentially introduced into pilot seed tanks to build a massive, highly active vegetative biomass before inoculation into the main fermenter.

Carbon & Energy Source: Glucose or sucrose is added for initial rapid growth. Lactose is added as a slowly metabolizable sugar that maintains the prolonged idiophase (the phase where penicillin is produced).

Nitrogen & Amino Acid Source: Corn steep liquor (CSL) is heavily utilized because it provides organic nitrogen, amino acids, and minerals.

Mineral Salts: KH₂PO₄, MgSO₄, ZnSO₄, and MnSO₄.

Precursors: Specifically added to direct the synthesis toward the desired penicillin type. Phenylacetic acid or potassium phenylacetate is added to yield Penicillin G, while phenoxyacetic acid is added for Penicillin V.

Fermentation Conditions:

• Process Type: Fed-batch fermentation is used, where feeds of glucose and precursors are controlled continuously to prevent catabolite repression.
• Temperature: Maintained closely between 24-26°C.
• pH: Kept around 6.5 initially, and not allowed to exceed 7.0-7.5 to maintain penicillin stability. Acid or ammonia is added automatically to regulate pH fluctuations.
• Aeration & Agitation: High oxygen supply is vital. Sterile air is introduced under pressure through a sparger alongside aggressive impeller agitation.
• Duration: The fermentation process lasts for 6-7 days.

Hybridoma Technology and Monoclonal Antibodies

Monoclonal Antibodies (mAbs): Highly uniform, homogeneous antibodies engineered by an identical clone of immune cells. They possess monovalent affinity, meaning they bind to one identical, specific epitope on an antigen target.

Hybridoma Technology: Developed by Georges Köhler and César Milstein in 1975 (which earned them a Nobel Prize). The methodology involves fusing short-lived, antibody-secreting B-lymphocytes with immortal cancerous myeloma cells to create a hybrid cell line called a hybridoma. Hybridomas carry both key characteristics: specific antibody production and infinite replication capability.

Step-by-Step Procedure:

1. Immunization: A mouse is injected with a targeted antigen to trigger an immune response, causing its B-cells to start synthesizing specific corresponding antibodies.
2. Isolation of Splenocytes: The mouse is later sacrificed, its spleen is harvested, and antibody-producing B-lymphocytes are extracted.
3. Preparation of Myeloma Cells: Special cancerous B-cells (myeloma cells) that lack the enzyme HGPRT (Hypoxanthine-Guanine Phosphoribosyltransferase) are cultured. Because they are HGPRT-deficient, these cells cannot use the salvage pathway to synthesize DNA.
4. Cell Fusion: The isolated B-lymphocytes and the HGPRT-deficient myeloma cells are mixed together and fused using Polyethylene Glycol (PEG) or electrofusion to produce hybrid cells.
5. Selection in HAT Medium: The heterogeneous mixture containing fused hybridomas, unfused B-cells, and unfused myeloma cells is introduced into HAT Medium (Hypoxanthine, Aminopterin, and Thymidine).

Applications of Monoclonal Antibodies:

• Diagnostic Applications: Used widely in commercial home pregnancy test kits (to identify hCG levels), blood typing, tissue typing for organ transplants, and identifying specific viral/bacterial infections or cancer biomarkers.
• Therapeutic Applications: Utilized as targeted "magic bullets" for oncology treatments (e.g., Rituximab for Non-Hodgkin lymphoma, Trastuzumab for breast cancer) and treating autoimmune/inflammatory diseases (e.g., Infliximab for Crohn's disease and rheumatoid arthritis).

Processing and Storage of Human Plasma Fractions

Introduction: Human blood plasma holds an array of clinically vital proteins. Instead of administering whole plasma, these components are isolated into purified concentrates through chemical fractionation.

Cohn Fractionation Method: Developed by Edwin Cohn during WWII, this standard process uses cold ethanol fractionation. By altering ethanol concentration, temperature, pH, ionic strength, and protein concentration step-by-step, major plasma proteins are sequentially precipitated out from the mixture.