DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY AND BIOTECHNOLOGY
PHARMACEUTICAL MICROBIOLOGY FOR 500L: PMB 573
TOPIC: BIOTECHNOLOGICAL PRODUCTS AND PHARMACEUTICAL CARE
Pharm. Oluebube. O
LECTURE OUTLINE
● Introduction
● An overview of relevant information service to patient on: Storage, Reconstitution,
Stability, Antigenicity and Self-administration
INTRODUCTION
Biotechnological products in pharmaceutical care are made using biological organisms produced
using biotechnology (genetic engineering, recombinant DNA technology, and cell culture
techniques). This product can be new or modified products, methods and organisms intended to
improve human health and society. They are applicable in modern therapeutics and in the
treatment of diseases.
Importance of biotechnology in modern pharmaceutical care
● Biotechnology enables the creation of new drugs
● used to produce therapeutic proteins, antibodies, and vaccines, which are essential for
treating various conditions like cancer, autoimmune disorders, and infectious diseases.
● Biotechnology has revolutionized diagnostic capabilities through the development of
biomarkers, genetic testing, and molecular diagnostics, enabling early diagnosis and
personalized medicine.
● These innovations in drug development, diagnosis, and treatment had led to improved
patient outcomes and enhanced healthcare.
There are various biotechnological products used in pharmaceutical care
Product Type Example Main Use
Recombinant Insulin Insulin glargine/lispro Diabetes
Monoclonal Antibodies Trastuzumab, Rituximab Cancer, autoimmune diseases
Erythropoietin Epoetin alfa Anemia, cancer
Vaccines Pfizer-BioNTech, HPV, Hep Infectious disease prevention
(mRNA/Recombinant) B
Growth Hormone Somatropin Growth hormone deficiency
Biosimilars Trastuzumab biosimilars Cheaper access to biologics
OVERVIEW OF BIOTECHNOLOGICAL PRODUCT TYPES WITH RELEVANT INFORMATION SERVICE
TO PATIENT
1. Recombinant proteins and hormones eg insulin, erythropoietin
Recombinant proteins are biologically active proteins artificially produced through recombinant
DNA (rDNA) technology a process where genes are artificially inserted into microorganisms (like
bacteria or yeast) to produce proteins. These proteins have undergone advancements over time,
with improvements in their structures, properties, and administration routes, resulting in higher
efficiency and increased safety.
Steps in Production of Recombinant Proteins: insulin
● Isolate the human insulin gene: Scientists extract the DNA containing the gene for human
insulin from human pancreatic cells.
● Construct the plasmid vector: A small, circular DNA molecule called a plasmid is obtained
from bacteria and modified to include the human insulin gene. The plasmid is an essential
tool used to deliver the human insulin gene into a host microorganism and can divide
independently.
● Insert the gene into the plasmid: The human insulin gene is cut and inserted into the
plasmid which acts as a vector using specialized enzymes and DNA ligase.
● Transform bacteria: The recombinant plasmid is introduced into bacterial cells, typically
E. coli for insulin (Yeast or mammalian cells for complex proteins like epoetin alpha (EPO),
through a process called transformation.
● Fermentation and insulin production: The transformed bacteria are grown in large
fermentation tanks, where they replicate and produce human insulin based on the
inserted gene.
● Harvest and purification: The insulin produced by the bacteria is harvested from the
fermentation broth. This is done through centrifugation or filtration methods.
● Purification: The insulin is purified using various techniques, including chromatography,
ultrafiltration, precipitation to remove impurities and other proteins.
● Final product: The purified recombinant insulin is then formulated into a medicine for use
in treating diabetes.
Storage
Insulin: insulin should be stored in a refrigerator at a temperature between 2°C and 8°C (36°F to
46°F). it is stable at room temp for 4-6 weeks after opening.
EPO: needs a Strict cold chain storage (2–8°C) but should not be freeze. It is More temperature-
sensitive.
Both are protein-based and denature if exposed to heat or freezing.
Reconstitution
Insulin: Usually is a pre-mixed liquid needing no reconstitution.
EPO: Usually is a ready-to-use liquid however some formulations may require gentle mixing and
not shaking. This is because some liquid biologics may settle (e.g., particles or proteins may
distribute unevenly), they need to be mixed gently. Shaking vigorously can; Damage the protein
structure leading to the inability of the insulin to bind to its receptor effectively. Shaking can also
cause clumping, foaming, Bubble formation and reduce the drug's effectiveness or Increase risk
of immune reactions (due to altered protein)
Note that Reconstitution requirements vary by brand and formulation.
Stability
Proteins generally can be degraded by heat, light, agitation. Hence stability can be extended by
the use of Refrigeration, Use of stabilizers, proper packages
Antigenicity: refers to a substance's ability to be recognized by the immune system and trigger
an immune response.
Antigenicity is rare in human Insulin but can occur when the protein nature is altered or the drug
contains contaminants or impurities. This can be avoided by using Humanized or fully human
proteins, Use of pegylation (PEG-modification to mask proteins), ensuring impurities are avoided
in preparation and administration.
Administration
Insulin is Administered via subcutaneous injection (pens, syringes, pumps) and so Patients often
trained to self-inject.
EPO is Administered IV in dialysis or subcutaneously in outpatient care
2. Monoclonal antibodies (e.g., trastuzumab, adalimumab)
Monoclonal antibodies (mAbs) are large, Y-shaped immune system proteins designed to
recognize and bind to specific targets (antigens).
Steps in production
● Immunization: A mouse (or other animal) is injected with the antigen of interest eg
Human Epidermal Growth Factor Receptor 2 (HER2) in breast cancer, which triggers the
production of antibodies specific to that antigen by B cells.
● B-cell Collection: B cells from the immunized animal's spleen or lymph nodes are
harvested. We harvest the B-cells (beta cells) — not the antibodies — because B-cells are
the source of the antibodies and can be fused with myeloma cells to create immortal
hybridomas that will continuously produce the specific antibody.
● Fusion with Myeloma Cells: These B cells are fused with myeloma cells (cancerous B cells
that can divide indefinitely) using a chemical or electrical fusion method. This fusion
creates hybridomas, which inherit both the antibody-producing ability of the B cell and
the immortality of the myeloma cell.
● Selection and Cloning: Hybridomas are screened (using ELISA, Western blot, or flow
cytometry) to identify those that produce the desired antibody. Recall that when beta
cells where harvested both cells specific for the given antigen and those not specific
where used, hence not all the hybridoma will produce HER2 specific antibodies, hence
the need for selection. Selected hybridomas are then cloned to create a stable cell line
that can produce the monoclonal Ab indefinitely.
● Antibody Production: The cloned hybridoma cell lines are grown in culture (either in vivo
or in vitro) to produce large quantities of the monoclonal antibody.
● Purification: The mAb is then purified from the culture medium using various techniques
to isolate it.
Storage
Refrigeration (2–8°C) required for both. Do not freeze
Reconstitution
Trastuzumab (IV form) comes in form of lyophilized powder to be reconstituted with sterile
water. It can also come as a subcutaneous inject which does not need reconstitution.
Adalimumab comes as a subcutaneous injection. it is important not to shake these products
vigorously to avoid altering their structural nature.
Stability
Monoclonal antibodies are sensitive to Heat, Freezing, Light and Agitation. Once reconstituted
(for IV), use within specific time window (e.g., 24 hours refrigerated).
Antigenicity
● Immune response may be triggered when non-human sequences remain in the protein
● Impurities or aggregates are present
● Dosing is intermittent or irregular
● Risk of antigenicity is higher in Trastuzumab and lower in Adalimumab. Why ?
Trastuzumab is a humanized monoclonal antibody meaning only the antigen-binding
regions (CDRs) from a mouse antibody are grafted onto a human. Though mostly human,
the mouse-derived regions can still be recognized as foreign, especially in long-term
treatment.
Adalimumab is a fully human monoclonal antibody the antibody: it is 100% human in
structure, minimizing immune reactions (antigenicity) when administered to patients.
One method used here is the genetically engineered transgenic mice that have had their
mouse antibody genes replaced with human antibody genes
Self-Administration
This is only appropriate for subcutaneous inject when patient have been trained.
3. Vaccines (e.g., mRNA, recombinant HPV vaccine)
Vaccines are biological preparations designed to prevent infectious diseases by stimulating the
immune system to recognize and fight pathogens (like viruses or bacteria) without causing illness.
Type What It Uses Example
Inactivated Killed virus or bacteria Hepatitis A, polio (IPV)
Live attenuated Weakened form of the virus MMR, yellow fever
Subunit / Protein- Specific parts that stimulate immune HPV, hepatitis B
based response (e.g., proteins, VLPs)
Toxoid Inactivated toxins Tetanus, diphtheria
Mrna Genetic code that instructs cells to make a Pfizer COVID-19
harmless viral protein
Viral vector Harmless virus carrying a gene from the Oxford-AstraZeneca
target pathogen COVID-19
Vaccine production stages
Vaccine production involves several key stages. This manufacturing process includes:
● Antigen preparation (inactivation/attenuation): This stage focuses on generating the
antigen component of the vaccine. This may involve inactivating/killing the pathogen
(e.g., Hepatitis A, flu), weakening it, or producing specific antigenic components.
● Purification: The prepared antigen is then purified to remove unwanted materials and
ensure its quality and safety.
● Formulation: The purified antigen is combined with other ingredients, such as adjuvants
(to enhance the immune response), stabilizers (to maintain vaccine potency), and
preservatives (to prevent bacterial contamination), to create the final vaccine
preparation.
Steps in production of mRNA
An mRNA stands for messenger Ribonucleic Acid. It’s like a set of instructions or a recipe your
body uses to make proteins.
An mRNA vaccine delivers a synthetic messenger RNA into the body to instruct cells to produce
a viral protein (e.g., spike protein) — which triggers the immune system to make antibodies.
● Gene Selection: Scientists first identify the genetic sequence for a viral protein that
triggers immunity, it does not contain the virus and so cannot cause virus — for example,
the spike protein of the COVID-19 virus.
● In Vitro Transcription: A DNA template containing that viral gene is used in the lab to
produce mRNA through a process called transcription. This is done without using living
cells — it's a completely synthetic process.
● mRNA Modification: The mRNA is then chemically modified (such as adding a 5' cap, a
poly-A tail, or modified nucleosides) to improve its stability and reduce the chance of
being destroyed by the body’s immune system before it works.
● Encapsulation: The fragile mRNA is packaged inside lipid nanoparticles (tiny fat-like
droplets) to protect it and help it enter human cells efficiently.
● Formulation and Freezing The final vaccine is formulated with stabilizers and stored in
ultra-cold conditions to preserve its integrity until it’s ready for use.
When such vaccine is injected into the body,
1. The mRNA enters the cell but stays in the cytoplasm (it does NOT enter the nucleus or
affect DNA).
2. Ribosomes (the cell’s protein factories) read the mRNA instructions and makes the viral
protein, in this case spike protein against COVID (just the protein, not the whole virus
3. The viral protein is displayed on the cell surface or released into the body. The immune
system recognizes it as foreign and starts an immune response.
4. The immune system now "remembers" the spike protein If the real virus later enters the
body.
Storage
mRNA vaccines are very temperature-sensitive. Some require ultra-cold storage (–80°C to –60°C),
while newer formulations can be stored at standard refrigerator temperatures (2–8°C) for a
limited time. Repeated thawing and freezing must be avoided.
Reconstitution
Some versions need to be diluted before use (e.g., with sterile saline), while others come ready
to use. Healthcare providers must check the manufacturer’s instructions.
Stability
Once thawed and/or reconstituted, mRNA vaccines have limited stability. They are usually only
stable for a few hours to a few days depending on storage conditions. They must be protected
from light and not shaken vigorously.
Antigenicity
Since mRNA vaccines do not contain the actual virus or protein, the risk of causing an unwanted
immune response (antigenicity) is very low. However, immune reactions like soreness, fever, and
fatigue are common due to immune activation.
Administration
Administered as an intramuscular injection. Most require multiple doses (e.g., an initial dose and
one or more boosters).
Recombinant protein vaccines (e.g., HPV Vaccine – Gardasil)
These vaccines use a purified viral protein, produced in a lab using recombinant DNA technology,
to stimulate the immune system. They do not contain any live virus, which makes them safer,
especially for immunocompromised individuals.
Steps in production
● Gene Cloning: The gene that codes for the target viral protein (e.g., the L1 protein of HPV)
is identified and inserted into a circular DNA molecule called a plasmid. This plasmid is
then introduced into a host cell, typically yeast (e.g., Saccharomyces cerevisiae) or insect
cells.
● Protein Expression: The host cells, now containing the inserted gene, are cultured in
bioreactors. These cells read the inserted gene and begin producing virus-like particles
(VLPs). VLPs mimic the structure of the virus but do not contain any genetic material, so
they cannot replicate or cause infection.
● Purification: The VLPs are harvested from the culture and purified using processes such
as filtration and chromatography. This removes any unwanted cellular material, ensuring
a clean and concentrated vaccine component.
● Formulation with Adjuvant: The purified VLPs are mixed with an adjuvant, often
aluminum salts, which help to enhance the body’s immune response to the vaccine. This
makes the vaccine more effective at producing lasting immunity.
● Packaging: The final vaccine formulation is carefully filled into sterile vials or prefilled
syringes, sealed, and labeled under strict quality control conditions. It is then stored and
distributed under proper cold chain conditions until use.
Storage
These vaccines are generally more stable than mRNA vaccines. They are stored in a refrigerator
at 2–8°C. Freezing should be avoided to prevent damage to the protein components.
Reconstitution
Most recombinant protein vaccines are provided in ready-to-use liquid form and do not require
reconstitution.
Stability
They are relatively stable under proper refrigeration and can tolerate short-term exposure to
room temperature, although prolonged exposure may reduce potency.
Antigenicity
Since these vaccines contain non-infectious protein particles (like virus-like particles), they have
a low risk of causing severe immune reactions. Local inflammation or mild fever can occur due to
the immune response or adjuvants added to enhance effectiveness.
Administration
Given via intramuscular injection. Multiple doses may be required, usually spaced weeks to
months apart, depending on age and immunization history.
4. Gene and cell therapies (brief mention)
Gene Therapy
Gene therapy involves introducing, removing, or altering genetic material (DNA or RNA) within
a person’s cells to treat or prevent disease. Used to replace faulty genes, silence harmful ones,
or insert new genes. Instead of using drugs or surgery, gene therapy targets the root cause of a
disease (the genes) to replace a mutated or faulty gene with a healthy copy. Often delivered using
viral vectors (like AAV, lentivirus) or non-viral methods (lipid nanoparticles, CRISPR).
Cell Therapy
Cell therapy involves transplanting or injecting live cells into a patient to repair, replace, or
enhance cellular function. Cells may be from the patient (autologous) or a donor (allogeneic).
Includes stem cells, immune cells, or gene-modified cells (like CAR-T cells).
Gene Therapy Production Steps
● Gene Identification – A functional version of the target gene is selected or synthesized.
● Vector Design – The gene is inserted into a viral or synthetic vector.
● Vector Production – Engineered viruses are grown in special cell lines, could be bacteria
or human. It is cultured in a large scale and then purified.
● Formulation – The gene vector is stabilized and packaged (as a solution for injection).
● Sterility & Testing – Product is tested for purity, potency, and sterility before release.
Cell Therapy Production Steps
● Cell Collection – Cells are collected from the patient or donor (e.g., blood or bone
marrow).
● Cell Engineering/Expansion – In the lab, cells are:
1. Genetically modified (if needed)
2. Stimulated to grow and multiply
● Purification & Testing – Cells are purified, tested for viability and function.
● Cryopreservation – Final cell product is frozen for transport or storage.
● Thawing & Infusion – Just before use, the cells are thawed (warming previously
cryopreserved or frozen cells to restore their viability and functionality for use in
therapeutic application or further manufacturing steps) and infused into the patient.
Key Pharmaceutical Care Properties of gene and cell therapies
Storage
● Gene therapies: Often stored refrigerated or frozen (–20°C to –80°C); lipid nanoparticle
formulations (e.g., mRNA vectors) may require ultra-cold conditions.
● Cell therapies: Stored in liquid nitrogen (–150°C or below) to maintain cell viability.
Reconstitution
● Gene therapies: Some products may need mixing with a diluent before administration.
● Cell therapies: Usually require thawing of cryopreserved bags under controlled
conditions. No further reconstitution, but must be infused quickly after thawing.
Stability
● Highly sensitive: Once reconstituted or thawed, stability is limited (often just hours).
● Degradation can occur with temperature fluctuations, mechanical stress, or time.
Antigenicity
● Gene therapy: Risk of immune reaction to viral vectors (especially with prior exposure).
● Cell therapy: Risk of graft-versus-host disease (for allogeneic cells) or cytokine release
syndrome (especially in CAR-T therapy).
Graft-versus-Host Disease (GVHD) is a serious immune complication that can occur after
allogeneic cell therapy or stem cell (bone marrow) transplantation, when immune cells
from a donor ("the graft") attack the recipient's body ("the host").
This can be averted by avoiding donor and host mismatches.
Also, the use of immunosuppressant.
Administration
● Typically administered intravenously (IV) in a hospital or specialized center.
● Gene therapy may be a one-time injection; cell therapy may require preconditioning
treatment (e.g., chemotherapy) before infusion.
Examples
● Gene therapy: Zolgensma – for spinal muscular atrophy
● Cell therapy: Kymriah (CAR-T therapy) – for leukemia; Stem cell transplants – for bone
marrow failure
Conclusion
The use of biotechnology has greatly improved modern therapeutics in both treatment and
prevention of diseases. However strict guidance most be ensured in their use to avoid adverse
effect. Patient, care givers and health practitioners need to be aware of this relevant information
on Storage, Reconstitution, Stability, Antigenicity and Self-administration
Sources
● Biopharmaceuticals: Biochemistry and Biotechnology by Gary Walsh
● Remington: The Science and Practice of Pharmacy by David B. Troy et al.
● Pharmaceutical Biotechnology: Concepts and Applications by Gary Walsh
