B Pharmacy Sem 6: Pharmaceutical Biotechnology
Delve into PCR, recombinant protein production, immunoassays, fermentation scale‑up, and blood‑derived therapeutics in Pharmaceutical Biotechnology
Subject 5: Pharmaceutical Biotechnology
Unit 1 – Introduction, Enzymes, Biosensors, Protein Engineering
Unit 2 – Genetic Engineering & Recombinant Technologies (PCR, vectors, insulin, vaccines, interferon)
Unit 3 – Immunotechniques & Microbial Products (ELISA, hybridoma, fermentation)
Unit 4 – Immunoassays & Microbial Genetics
Unit 5 – Fermentation Scale‑up & Blood Products
Unit 1: Introduction, Enzymes, Biosensors & Protein Engineering
This unit lays the foundation of biotechnology in pharmacy, covering basic concepts, enzyme applications, biosensor technology, and protein engineering strategies—presented for clear learning and exam readiness.
5.1 Introduction to Pharmaceutical Biotechnology
Definition & Scope
Use of biological systems, organisms, or derivatives to develop drugs and diagnostics.
Key applications: recombinant proteins, monoclonal antibodies, vaccines, gene therapy.
Historical Milestones
Insulin production via E. coli recombinant DNA (1982).
Development of monoclonal antibody technology (Köhler & Milstein, 1975).
Regulatory Considerations
Good Biotechnology Practices (GBP): parallels GMP but includes genetic containment, cell-bank management, and biosafety.
5.2 Enzymes in Biotechnology
Enzyme Classification (EC 1–6)
Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases.
Therapeutic & Industrial Uses
Therapeutic: Alteplase (tPA) for thrombosis; Asparaginase in leukemia.
Industrial: Immobilized enzymes in drug synthesis (e.g., lipases for chiral intermediates).
Immobilization Techniques
Adsorption, covalent binding, entrapment in gels → enhances stability and reusability.
5.3 Biosensors
Basic Components
Bioreceptor: Enzyme, antibody, nucleic acid that specifically binds analyte.
Transducer: Converts biorecognition event into a measurable signal (electrochemical, optical, piezoelectric).
Signal Processor: Amplifies and displays result.
Key Types & Examples
Glucometer: Glucose oxidase + electrochemical detector for blood sugar monitoring.
Immunosensor: Antibody-coated surface + optical transducer for drug/toxin detection.
Performance Parameters
Sensitivity, Specificity, Response Time, Stability.
5.4 Protein Engineering
Rational Design (Site‑Directed Mutagenesis)
Alter specific amino acids to improve stability, activity, or binding (e.g., PEGylation sites).
Directed Evolution
Generate libraries of gene variants → iterative rounds of mutation and selection for enhanced traits (e.g., thermostable enzymes).
Applications
Improved Biologics: Enhanced antibody affinity, reduced immunogenicity.
Novel Biocatalysts: Enzymes with tailored specificity for drug synthesis.
Key Exam Tips
Biotech definitions: Differentiate GMP vs. GBP and recall biotech milestones.
Enzyme uses: Know therapeutic vs. industrial applications and immobilization benefits.
Biosensor anatomy: Bioreceptor + transducer + processor; glucometer is a classic example.
Engineering strategies: Rational design is site-specific; directed evolution mimics natural selection in vitro.
Unit 2: Genetic Engineering & Recombinant Technologies
This unit delves into molecular biology tools and bioprocesses used to produce therapeutic proteins, vaccines, and diagnostics, focusing on PCR, vectors, and key recombinant products like insulin and interferon.
5.2.1 Polymerase Chain Reaction (PCR)
Definition: Enzymatic amplification of specific DNA sequences.
Key Steps:
Denaturation (94–98 °C): Separates DNA strands
Annealing (50–65 °C): Primers bind target sequence
Extension (72 °C): Taq polymerase synthesizes new strands
Applications: Gene cloning, mutation analysis, diagnostics (e.g., SARS‑CoV‑2 detection).
5.2.2 Vectors for Gene Expression
Plasmid Vectors: Circular DNA for bacterial expression (e.g., pBR322, pUC series).
Bacteriophage Vectors: Higher capacity; λ phage for genomic libraries.
Viral Vectors:
Adenoviral, Retroviral, Lentiviral vectors for gene therapy and vaccine delivery.
Advantages: High transduction efficiency; Limitations: Immunogenicity, insertional mutagenesis.
Key Features: Origin of replication, selectable marker (antibiotic resistance), multiple cloning site, promoter.
5.2.3 Recombinant Protein Production
Host Systems:
Bacterial (E. coli): Rapid growth, high yield; lacks post‑translational modifications.
Yeast (S. cerevisiae): Some glycosylation; easy scale‑up.
Mammalian Cells (CHO, HEK293): Authentic folding & glycosylation; lower yield, higher cost.
Process Flow: Gene insertion → transformation/transfection → expression → downstream purification (chromatography).
Purification Tags: His‑tag, GST‑tag for affinity chromatography.
5.2.4 Recombinant Therapeutics
Insulin
History: First recombinant human insulin in E. coli (1982).
Formats: Regular, NPH (isophane), analogs (lispro, glargine) for tailored PK profiles.
Interferons
Types: IFN‑α (viral hepatitis, malignancies), IFN‑β (multiple sclerosis), IFN‑γ (chronic granulomatous disease).
Production: E. coli or yeast expression; PEGylation for extended half‑life.
Vaccines
Subunit & Conjugate: Recombinant hepatitis B surface antigen in yeast.
Viral‑Vector: Ebola and COVID‑19 adenoviral vaccines.
Key Exam Tips
PCR conditions: Denature, anneal, extend—know temperatures and purpose.
Vector elements: Origin, marker, MCS, promoter—critical for expression.
Host choice: Balance yield vs. post‑translational needs.
Recombinant products: Remember first recombinant insulin and PEGylated interferons.
Unit 3: Immunotechniques & Microbial Products
This unit covers immunological assays for diagnostics and microbial fermentation for product synthesis. Focus is on ELISA, hybridoma technology, and fermentation processes with clear definitions and exam‑ready detail.
5.3.1 Enzyme‑Linked Immunosorbent Assay (ELISA)
Definition: A plate‑based assay that uses enzyme‑conjugated antibodies to detect and quantify antigens or antibodies.
Formats:
Direct ELISA: Antigen adsorbed → enzyme‑labeled antibody binds directly → substrate conversion measured.
Indirect ELISA: Antigen adsorbed → primary antibody binds → enzyme‑labeled secondary antibody binds primary.
Sandwich ELISA: Capture antibody immobilized → antigen binds → detection antibody (enzyme‑linked) completes the “sandwich.”
Competitive ELISA: Sample antigen competes with labeled antigen for limited antibody binding sites.
Key Steps: Coating ⁞ blocking ⁞ incubation with antibody ⁞ substrate reaction ⁞ readout (OD).
Applications: Hormone levels (e.g., insulin), infection markers (e.g., HIV antibodies), cytokine quantification.
5.3.2 Hybridoma Technology for Monoclonal Antibodies
Definition: Production of monoclonal antibodies (mAbs) by fusing a specific antibody‑producing B cell with an immortal myeloma cell.
Process Flow:
Immunization: Animal (usually mouse) injected with antigen → B cells activated in spleen.
Fusion: Spleen B cells fused with myeloma cells using polyethylene glycol → hybridomas.
Selection: Hybridomas grown in HAT medium (selects fused cells).
Screening & Cloning: Identify clones producing the desired antibody → expand for mAb production.
Advantages: Infinite supply of uniform, high‑affinity antibodies.
Applications: Therapeutics (e.g., rituximab), diagnostics (e.g., pregnancy tests), research.
5.3.3 Microbial Fermentation & Products
Definition: Cultivation of microorganisms under controlled conditions to produce valuable biochemicals.
Types of Fermentation:
Batch: All nutrients added at start; culture allowed to grow until nutrients depleted.
Fed‑Batch: Nutrients added intermittently to extend productive phase.
Continuous (Chemostat): Fresh medium added and culture removed continuously to maintain steady state.
Key Parameters: pH, temperature, dissolved oxygen, agitation rate, nutrient concentration.
Major Products:
Antibiotics: Penicillins (Penicillium chrysogenum), cephalosporins.
Amino Acids: Lysine, glutamic acid (Corynebacterium glutamicum).
Organic Acids & Enzymes: Citric acid (Aspergillus niger), proteases, amylases.
Downstream Processing: Cell separation (centrifugation/filtration) → product recovery (precipitation, chromatography) → purification → formulation.
Key Exam Tips
ELISA formats: Remember sandwich = two antibodies; direct = one antibody.
Hybridoma: HAT medium selects for fused cells; fusion yields infinite monoclonal source.
Fermentation modes: Batch (simple), fed‑batch (extended), continuous (steady‑state).
Microbial products: Link each microbe to its product (e.g., Penicillium → penicillin).
Unit 4: Immunoassays & Microbial Genetics
This unit examines advanced immunoassay techniques for detection and quantification, and the genetic tools used to engineer microbial strains for enhanced bioproduct synthesis—presented with precise definitions and exam‑ready clarity.
5.4.1 Advanced Immunoassays
Radioimmunoassay (RIA)
Definition: Uses radioactive isotopes (e.g., I‑125) labeled to antigen or antibody to quantify analytes via competitive binding.
Key Feature: High sensitivity (picogram range); requires radiation safety.
Application: Hormone levels (TSH, insulin), drug monitoring.
Fluorescence Immunoassay (FIA)
Definition: Employs fluorophore‑labeled antibodies; fluorescence intensity proportional to analyte concentration.
Advantage: No radiation hazard; multiplexing possible.
Application: Allergy testing, viral antigen detection.
Chemiluminescent Immunoassay (CLIA)
Definition: Labels antibodies with chemiluminescent substrates; light emission measured by luminometer.
Benefit: Very high sensitivity, wide dynamic range.
Application: Cardiac markers (troponin), infectious disease serology.
5.4.2 Microbial Genetics
Mutagenesis Techniques
Physical Mutagenesis: UV irradiation causes thymine dimers → random mutations.
Chemical Mutagenesis: Agents (e.g., nitrosoguanidine) induce point mutations.
Site‑Directed Mutagenesis: Precise base changes using PCR‑based methods to alter specific amino acids in enzymes.
Recombinant Strain Development
Gene Knockout: Deletion of undesirable genes (e.g., proteases that degrade product).
Gene Overexpression: Inserted under strong promoters to boost product‑pathway enzymes.
Plasmid Curing & Stability: Ensuring maintenance of expression plasmids without antibiotic pressure (e.g., toxin–antitoxin systems).
Marker Rescue & Selection
Auxotrophic Markers: Complementation of a nutritional deficiency (e.g., His⁻ → His⁺) for selection.
Antibiotic Resistance Markers: Traditional, but raise biosafety concerns.
Genome Editing Tools
CRISPR‑Cas Systems: Targeted double‑strand breaks and repair enable precise edits (knock‑in/knock‑out).
Recombineering: Phage‑derived recombination proteins facilitate insertion/deletion with linear DNA.
Key Exam Tips
Immunoassays: RIA = radioactive; FIA = fluorescent; CLIA = chemiluminescent—rank by safety and sensitivity.
Mutagenesis: Distinguish random (UV/chemical) vs. targeted (site‑directed).
Strain engineering: Know knockout vs. overexpression strategies and selection markers.
Genome editing: CRISPR‑Cas offers highest precision; recombineering for bacteria.
Unit 5: Fermentation Scale‑Up & Blood Products
This unit addresses the scale‑up of microbial fermentation processes for commercial production and the preparation and use of blood‑derived therapeutic products, with concise definitions for exam readiness.
5.5.1 Fermentation Scale‑Up
Scale‑Up Principles
Objective: Transition from laboratory to industrial scale while maintaining yield and product quality.
Key Considerations:
Geometric Similarity: Maintain aspect ratios (e.g., height:diameter) of vessels.
Kinetic Similarity: Match key dimensionless numbers (Reynolds, Péclet) to preserve mixing and mass transfer.
Physicochemical Parameters: pH, temperature, dissolved oxygen (DO), shear stress.
Bioreactor Types
Stirred‑Tank Reactor (STR): Most common; mechanical agitation for mixing and oxygen transfer.
Airlift Reactor: Gas-driven circulation; gentler shear for shear‑sensitive cells.
Packed‑Bed & Fluidized‑Bed: Immobilized cells on support matrices; continuous operation.
Scale‑Up Strategies
Constant Tip Speed: Keep impeller tip speed the same to control shear.
Constant Power Input per Volume (P/V): Maintain energy dissipation for mixing.
Constant k_La (Volumetric O₂ Transfer Coefficient): Ensure oxygen supply meets cell demand.
Downstream Processing (DSP)
Steps: Cell removal (centrifugation/filtration) → product concentration (ultrafiltration) → purification (chromatography, precipitation) → formulation.
Focus: Minimize product degradation; maximize purity and yield.
5.5.2 Blood‑Derived Therapeutic Products
Source Material & Collection
Whole Blood: Collected with anticoagulant; separated into components.
Plasma Apheresis: Continuous collection of plasma, return of cellular components.
Major Products & Definitions
Albumin
Definition: Plasma protein (~60 % of total), maintains oncotic pressure.
Use: Hypovolemia, burns, hypoalbuminemia.
Processing: Cold ethanol fractionation (Cohn method), pasteurization for viral inactivation.
Immunoglobulins (IVIG)
Definition: Pooled IgG from healthy donors.
Use: Primary immunodeficiency, autoimmune diseases (ITP).
Processing: Cold ethanol fractionation → chromatography → viral clearance (solvent/detergent).
Coagulation Factors
Examples: Factor VIII (hemophilia A), Factor IX (hemophilia B).
Processing: Cryoprecipitation of plasma → factor concentration → viral inactivation.
Clotting Components
Fresh Frozen Plasma (FFP): Contains all clotting factors for coagulopathy.
Cryoprecipitate: Rich in fibrinogen, vWF, Factor VIII.
Safety & Quality Controls
Pathogen Inactivation: Heat, solvent/detergent, UV irradiation.
Viral Testing: ELISA/PCR for HIV, HBV, HCV.
Sterility & Endotoxin Testing: USP <71> sterility, LAL assay for endotoxin.
Key Exam Tips
Scale‑Up: Remember geometric, kinetic, and k_La similarity principles.
Bioreactors: STR for versatility; airlift for low shear; packed‑bed for immobilized systems.
Blood Products: Cohn fractionation yields albumin, immunoglobulins, factors; know uses of each.
Safety: Pathogen reduction and rigorous testing ensure therapeutic safety.