Pharmaceutical Biotechnology: An Overview

Pharmaceutical Biotechnology

• Pharmaceutical biotechnology uses microorganisms, plants and animals to produce useful compounds
• Pharmaceutical biotechnology is focused on biotechnological manufacturing of pharmaceutical products

Biotechnology in Ancient Times

• Historically, biotechnology relied on experience without understanding underlying principles
• Example: winemaking, was based purely on experience, not knowledge
• In 1870, Pasteur showed chemical conversions were performed by living cells
• Pasteur defined biochemical conversions as traditional processes

Biotechnology as a Science

• Biotechnological knowledge grew after Pasteur's discovery
• Specifically, the catalytic role of enzymes became apparent for biochemical conversions
• Biotech transitioned into a science when tools became available to control and optimize traditional processes
• Catalysis provides an alternative, fast reaction pathway
• Catalysis produces more stable products than the starting material with a lower activation energy
• Example: The enzyme peroxidase causes hydrogen peroxide to turn to water and oxygen, following the equation: 2 H2O2 → 2 H2O + O2

Molecular Biology

• Molecular biology caused an important breakthrough after its development
• In 1950, pioneers in molecular biology discovered that DNA encodes proteins
• Molecular biology controls all cellular processes
• The application of molecular biology was the catalyst for a new phase in biotechnology

Recombinant DNA Technology

• The development of recombinant DNA technology in the 70s allowed control of gene expression in organisms used for biotechnological manufacturing
• These technologies created ways to introduce foreign DNA into organisms
• Genetically modified organisms then created new biotech possibilities
• Molecular biotechnology relies on thorough DNA molecule knowledge and DNA manipulation technologies

Public Debate around Biotechnology

• Questions were raised about the potential risks of genetically modified organisms in production facilities
• Ethics were questioned surrounding the modification of genetic structures of living organisms

Gene Expression

• Genetic information is chemically encoded in DNA structure
• This DNA is passed to daughter cells through DNA replication during cell division
• During transcription DNA converts to RNA
• During translation RNA converts to protein
• The series of events can be summarized as: DNA -> RNA -> Protein

Biopharmaceutical Drugs

• Conventional pharmaceutical formulations use simple molecules
• These molecules treat symptoms using trial and error
• Biopharmaceuticals are complex biological molecules/proteins
• Biopharmaceuticals aim to eliminate the underlying mechanisms of a disease
• Pharmaceutical biotechnology creates complex, larger molecules using living cells
• These molecules are similar to those found in human cell, like bacterial, plant, animal or yeast cells
• Large pharmaceutical biotechnology molecules are injected whereas smaller molecules are delivered in tablet form

Benefits of Combining Pharmaceuticals and Biotechnology

• Combining pharmaceuticals and biotechnology leads to many advantages for human health
• Biopharmaceutical drugs design and produce drugs adapted to a person’s genetic makeup
• Pharmaceutical biotechnology companies develop tailor-made medicines for maximum therapeutic effects
• Pharmaceutical biotechnology leads to better vaccines
• Biotech companies design and produce safer vaccines using genetically-engineered organisms
• Vaccines created with pharmaceutical biotechnology minimise the risks of infection associated with conventional vaccines

Pharmaceutical Biotechnology Products

• Pharmaceutical biotechnology products include antibodies, proteins and recombinant DNA products
• Antibodies: are proteins produced by white blood cells to identify and fight bacteria, viruses and foreign substances in the immune system
• Monoclonal antibodies: represent developments in pharmaceutical biotechnology
• Monoclonal antibodies are created by expression of a single beta lymphocyte
• All secreted antibody molecules derived from a single dividing parent beta lymphocyte are genetically identical

Proteins

• Proteins: are complex molecules, made of amino acids, doing most of the work inside the cells
• Proteins provide the structure, function, and regulation of tissues and organs
• Protein biotechnology is emerging as a key technology
• It will allow a better understanding of diseases like cancer or amyloid formation for better therapeutic intervention

Recombinant DNA Products

• Recombinant DNA: is the genetically engineered DNA created by recombining DNA fragments from different organisms
• Recombinant DNA products include:
  • Recombinant DNA vaccines
  • Recombinant DNA drugs
  • Recombinant DNA enzymes
  • Recombinant DNA growth hormones
  • Recombinant DNA insulin
  • Recombinant DNA proteins
  • Recombinant DNA yeast

Recombinant DNA Technologies

• Genetic modification of organisms fuses any DNA fragment to DNA molecules, which can maintain themselves
• These molecules can replicate independently
• These molecules, able to maintain themselves by autonomous replication, are called replicons

DNA Cloning Technology

• Recombinant DNA is a product of DNA cloning technology, in particular, through the use of plasmids
• Creating a recombinant DNA molecule is done by fusing a foreign DNA fragment to an isolated plasmid
• Replicons used as carriers for foreign DNA fragments are called vectors
• Vectors can include plasmids from bacteria or yeast or DNA from bactriovirus, animal or plant viruses
• Foreign DNA can be isolated from microbial, plant or animal cells
• Restriction enzymes cut DNA at specific sites
• Ligase enzymes close circular recombinant DNA
• Recombinant DNA introduction into a host cell forms a transformant
• Vectors replicate in the host, and all daughter cells inherit an exact copy (clone) of the recombinant DNA molecule

Monoclonal Antibodies

• Monoclonal antibodies are made by fusing myeloma cells with the spleen from a mouse that is immunized with the desired antigen

Microbial Considerations

• Proteins are administered parenterally, so sterility is essential
• Recombinant/purified protein vaccines include protein antigens like Hepatitis B vaccine I.M.
• Proteins are sensitive to other sterilization treatments, therefore can not withstand with autoclaving, gas sterilization or ionizing radiation
• Protein pharmaceuticals are assembled under aseptic conditions
• Assembly must then follow rules of the pharmaceutical industry for aseptic manufacture

Aseptic Manufacture

• Equipment and excipients are autoclaved, sterilised by dry heat (>160°C), chemical treatment or gamma radiation
• Filtration techniques used to remove micro-bacterial contaminants
  • Pre-filters remove the bulk of the bio-burden and other particulate materials.
  • The final 'sterilizing' filtration step passes through 0.2 or 0.22 µm membrane filters

Clean Rooms

• Products are made in class 100 clean rooms
• These rooms have a maximum of 100 particles per cubic foot (>0.5 µm)
• Class 100 rooms have laminar airflow and are filtered through HEPA (high efficiency particulate air) filters
• Cleanrooms maintain particulate-free air through HEPA or ULPA filters using laminar or turbulent airflow principles
• Air entering a cleanroom from outside is filtered to exclude dust
• Air is recirculated through HEPA and/or ULPA filters to remove internally generated contaminants
• Laminar airflow clean rooms use HEPA filters to filter and clean air entering the environment
• Laminar filters use stainless steel or other non-shedding materials to ensure a low particle count
• Filters make up roughly 80% of the ceiling space
• Cleanrooms with laminar airflow are called unidirectional airflow cleanrooms
• Non-unidirectional cleanrooms use turbulent airflow systems that clean particulate air
• Turbulent airflow filters create laminar flow and random, non-specific velocity
• Turbulent airflow can cause particles to move, which is difficult to separate from the air
• Therefore, non-unidirectional airflow systems depend non random movement to move particles to the filter

Considerations for Operators

• The 'human factor' is a major source of contamination
• Operators in facilities wear protective cloths (face masks, hats, gowns, gloves, head-to-toe overall garments)
• Critical factors for success are: regular filter exchange, validation of HEPA equipment and thorough cleaning of the room & equipment

Viral Decontamination

• Recombinant DNA products are grown in microorganisms
• Microorganisms should be tested for viral contaminations, and appropriate measures should be taken
• Removal of viral materials is done in the final product by filtration and then precipitation
• Viral contaminants in the final product are inactivated through heat or radiation
• Excipients with a risk factor, like blood-derived human serum albumin, should be tested before use

Pyrogen Removal

• Pyrogens are compounds that induce fever, with endogenous pyrogens and exogenous pyrogens representing the two types
• Exogenous pyrogens (pyrogens introduced into the body) derivates from bacteria, viral or fungal sources
• Bacterial pyrogens are mainly endotoxins shed from gram negative bacteria, also known as lipopolysaccharides (cell wall bacteria)
• Endotoxin pyrogens enter the bloodstream and bind to lipopolysaccharide binding PTNs
• Then bind to reticuloendothelial system (receptor cells of circulate Mononuclear and Polynuclear cells: CD-14 0f macrophages)
• This leads to the production and release of proinflammatory cytokines (IL-1 and IL-6), otherwise known as endogenous pyrogen
• In turn, this causes inflammation and fever by activating the arachidonic acid pathway.
• They aggregate and form large units with M.wt. of over 10^6 in water and sharing a general property of high negative electrical charge.
• Amphipathic compounds absorb to surfaces, therefore, hydrophillic and lipophillic endotoxins are lipopolysaccharides
• Endotoxins are stable during autoclaving, but break down with dry heat (treatment above 160°C for prolonged periods like, 30 minutes dry heat at 250°C).
• Pyrogen removal of recombinant products from bacterial sources is essential to prep
• Ion exchange chromatographic procedures can reduce endotoxin levels in solution
• Protein formulation excipients should be endotoxin-free
• Water for injection should be (freshly) distilled or produced by reverse osmosis
• Aggregated endotoxins cannot pass through the reverse osmosis membrane
• Removal of endotoxins can be done before filling the final container using activated charcoal or other materials with large hydrophobic surfaces
• Endotoxins can also be inactivated by oxidation (like peroxide) or dry heating (30 minutes dry heat at 250°C)