Recombinant Pharmaceuticals 1 and 2

Question 1

Define the following terms in Pharmaceutical Biotechnology

A) Biotechnology

B) Processes

C) Biopharmaceuticals

D) Pharmaceutical biotechnology

Answer 1

A)

• the use of living things, especially cells and bacteria, in industrial processes

B)

• genetic engineering

C)

• medicine and drugs that are produced using biotechnology

D)

• biotechnological manufacturing processes of pharmaceutical products

Question 2

What are the two types of cells?

Answer 2

• Prokaryotic cells
• Eukaryotic cells

Question 3

How are products of rDNA technology produced? Describe what are the ways. How is the desired product recovered?

Answer 3

Produced by genetic modification in which DNA coding for the required product is introduced

• by means of a plasmid or a viral vector into a suitable micro-organism or cell line, in which that DNA is expressed and translated into protein
• The desired product is then recovered by extraction and purification.
• The cell or micro-organism before harbouring the vector is referred to as the host cell, and the stable association of the two used in the manufacturing process is referred to as the host-vector system.

Question 4

Define what a vector and plasmid is.

Answer 4

Vector: A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism.

Plasmid: Plasmids are fragments of double-stranded DNA that typically carry genes and can replicate independently from chromosomal DNA

Question 5

What are the importance of plasmids?

Answer 5

• Easy to work with
• Self-replicating
• Stable
• Functional in many species and can be useful for a diverse set of applications

Question 6

How does human insulin production occur? (HUMULIN R)

Answer 6

HUMULIN R is produced by recombinant DNA technology utilising a non-pathogenic laboratory strain of Escherichia coli (E.coli)

• Human insulin producing gene taken out of DNA
• Plasmid DNA taken out of E.coli bacteria
• Bacterial DNA and human gene combined
• Plasmid is reintroduced into bacteria
• Engineered bacteria multiply producing insulin
• Insulin is separated and purified to produce human insulin

Question 7

What has to be considered when producing proteins for therapeutic use? What specifications have to be met?

Answer 7

A number of issues must be considered related to the manufacturing, purification and characterisation of the products

• Biotechnological products for therapeutic use have to meet strict specifications, especially when used via the parenteral route

Question 8

What are the two systems used to produce biotech products (proteins of therapeutic interest)? Give examples.

Answer 8

Expression systems

• Transgenic animals
• plants

Cultivation systems

• Medium –> contaminants

Question 9

Describe the process of how transgenic animals are used to get proteins of therapeutic interest.

Answer 9

• Foreign genes introduced into animals
• Desired protein is expressed in the milk

Advantage: Cheap method to produce in vast quantities

Disadvantage: Animal health

Question 10

Describe the process of how plants are used to get proteins of therapeutic interest.

Answer 10

• Human albumin has been expressed in potatoes

Issues: stability and phenolic oxidases

Potential: expressed protein in edible seeds eg rice

Question 11

For cultivation systems: medium;

A) What are the factors that have to be considered for the optimal growth of cells?

B) What else has to be considered?

C) How to prepare the final medium?

Answer 11

A)

• pH, oxygen tension, and temperature are chosen and controlled appropriately

B)

• medium with the proper nutrients is provided

C)

• components are dissolved in purified water before sterile filtration

Question 12

For cultivation systems: contaminants

A) What are examples of host-related contaminants?

B) What are examples of product-related contaminants?

C) What are examples of process-related contaminants?

Answer 12

A)

• Viruses
• Host derived proteins acid DNA
• Endotoxins (from gram-negative bacterial hosts)

B)

• Amino acid substitution and deletion
• Denatured protein
• Protein fragments
• Dimers and aggregates

C)

• Growth medium components
• Metals

Question 13

What is downstream processing? How does the purification cost compare to the upstream part of the production process?

Answer 13

Series of operations required to take biological materials such as cells, tissue culture fluid, or plant tissues, and derive from them a pure and homogeneous protein product.

• Recovering a biological reagent from a cell culture supernatant is one of the critical parts of the manufacturing procedure for biotech products and purification costs typically outweigh those of the upstream part of the production process

Question 14

What are the separation techniques involved in downstream processing? THREE different reasons.

Answer 14

Filtration/centrifugation

• Products from biotechnological industry must be separated from biological systems that contain suspended particulate material, including whole cells, lysed cell material, and fragments of broken cells
• the expense and effectiveness of such methods is highly dependent on the physical nature of the particulate material and of the product.

Precipitation

• The solubility of a particular protein depends on the physico-chemical environment, for example pH, ionic species and ionic strength of the solution

Chromatography

• Primarily separated based on differences in distribution between two phases, one which is the stationary phase (mostly a solid phase) and the other which moves
• Adsorption, Ion-exchange, immunoaffinity, hydrophobic interaction chromatographies

Question 15

What are the FOUR factors for formulating biotech products?

Answer 15

1. Excipients used in parenteral formulations of biotech products
2. Microbiological considerations
3. Shelf life of protein-based pharmaceuticals
4. Route of administration

Question 16

What are the excipients used in parenteral formulations of biotech products?

Answer 16

• Solubility enhancers
• Anti-adsorption and anti-aggregation agent
• Buffer components
• Preservatives and antioxidants
• Osmotic agents

Question 17

What microbiological considerations are there for the formulation of biotech products?

Answer 17

• Sterility
• Viral decontamination
• Pyrogen removal

Question 18

What has to be thought of for the shelf life of protein-based pharmaceuticals

Answer 18

Freeze drying of proteins

Question 19

What are the routes of administration for the formulation of biotech products?

Answer 19

• Parenteral route
• Oral

> protein degradation in the gastrointestinal (GI) tract and

> poor permeability of the wall of the GI tract in case of a passive transport process

> Oral vaccines

• Alternative routes (see attached image) –> nasal, pulmonary, rectal, buccal, transdermal

Question 20

What is blinatumomab used for? What does it bind? How is it made?

Answer 20

Anti-cancer therapy

> ndicated for the treatment of adults with acute lymphoblastic leukaemia (ALL)

• Blinatumomab is a bispecific T cell engager (BiTE®) antibody construct that selectively binds with high affinity to CD19 (expressed on cells of B-lineage origin) and CD3 (expressed on T cells)
• Using recombinant DNA technology, it is produced in a mammalian cell (Chinese hamster ovary) culture and is purified by a series of steps that include measures to inactivate and remove viruses

Question 21

What is gene therapy? What are the strategies of gene therapy?

Answer 21

Gene therapy is the use of nucleic acids as therapeutic medicinal compounds

Strategies

• Replacing a mutated gene that caused disease with a healthy copy of the gene
• Inactivating or ‘knocking out’ a mutated gene that is functioning improperly
• Introducing a new gene into the body to help fight a disease

Question 22

What are the two types of gene transfer?

Answer 22

Ex vivo versus in vivo gene therapy

In Vivo gene transfer: IV, IM, intra-arterial, intraperitoneal, intratumor or inhalation. Put gene therapy vector in the body.

Ex Vivo: take out donor cells from body, mix with gene therapy vector, grow it in culture, readminister via surgically implanted catheter

Question 23

What can gene therapy be used for?

Answer 23

• Gene therapy for Cancers

Correction of genetic mutations

• Gene therapy for vascular disease
• Gene therapy for monogenetic inherited disorders
• Gene therapy for infectious disease

Question 24

What is an example of gene therapy being used in cancer? How is it delivered?

Answer 24

Gendicine

• first-in-class gene therapy product to treat head and neck cancer
• Delivered via minimally invasive intratumoral injection, as well as by intracavity or intravascular infusion.

Question 25

What is used as gene therapy for sickle cell disease (monogenetic inherited disorders)?

Answer 25

hemoglobin-Beta gene found on chromosome 11

Question 26

What is the process of chimeric antigen receptor T-cell therapy (CAR-T therapy) --\> **immunotherapy**

Answer 26

1. **Extraction:** patients white blood cells separated from t cells. 2. **Reprogramming:** viral vector delivers a gene containing a chimeric antigen receptor (CAR) into the T cells 3. **Multiplication:** engineered cells now known as CAR T cells are multiplied in a bioreactor 4. **Preparation:** patient receives chemotherapy to lower their white blood cells and make room for incoming CAR T cells 5. **Treatment:** CAR T cells are infused into the patient's blood where they proliferate and detect and destroy cancer cells.

Question 27

What do CAR-T cells do?

Answer 27

Combines the specificity of a monoclonal antibody with the functions of a T cell in order to kill cancer cells.

Question 28

What are some challengees with CAR-T therapy?

Answer 28

* Takes time * Handling is difficult --\> T cells are sensitive and risk of cross-containation between patients * CAR-T cells are mainly for liquid tumours * Expensive

Question 29

What is another example of immunotherapy?

Answer 29

Dendritic cell-activated cytokine-induced killer cell-mediated (DC-CIK) immunotherapy * Ex vivo culture without genetically modifying the cells

Question 30

What are the examples of vectors for gene transfer? What are the best ones?

Answer 30

**Viral vectors** * Viruses, natural parasites that efficiently enter cellular targets and hijack cellular machinery for propagation, are currently the most effective vectors for gene therapy. * Approximately 70% of all gene therapy clinical trials employ viral vectors \> safety concerns

Question 31

What are examples of viral vectors for gene transfer?

Answer 31

* Retrovirus * Lentivirus * Adenovirus * Adeno-associated virus vectors * Poxvirus * Herpes simplex virus

Question 32

What are examples of non-viral vectors for gene transfer? What are the advantages and disadvantages of them?

Answer 32

* **Non-viral vectors generally consist of double-stranded recombinant DNA plasmids** alone or encapsulated in cationic polymer or lipidbased formulation **Advantages** * are generally non-immunogenic and can easily be readministered multiple times without causing a prohibitive immune response * have a reduced capacity for mutagenesis and a limited ability to produce unwanted by-products in vivo due to homologous recombination * Inexpensive to produce, especially on a large scale in contrast to viral vectors **Disadvantages** * low transduction efficiency * poor delivery to the therapeutic target * Cannot achieve sustained gene expression

Question 33

What is the purpose of vaccines? How do they work?

Answer 33

* To protect humans and domestic animals from infectious diseases \> the principle of vaccination is mimicking an infection in such a way that the natural specific defense mechanism of the host against the pathogen will be activated --\> host will remain free of the disease that normally results from a natural infection

Question 34

What are the properties that make an ideal vaccine?

Answer 34

* 100% efficient in all individuals of any age * Provides lifelong protection after single administration * Does not evoke any adverse reaction * Is stable under various conditions * Is easy to administer, preferably orally * Is cheap

Question 35

What is the BP definition for vaccines?

Answer 35

Vaccines for human use are preparations containing antigens capable of inducing a specific and active immunity against an infecting agent * Immune responses include the induction of the innate and the adaptive (cellular, humoral) parts of the immune system * Must show acceptable immunogenic activity and safety **Vaccines for human use may contain** * whole micro-organisms (bacteria, viruses or parasites) * antigens extracted from the micro-organisms or secreted by the micro-organisms or produced by genetic engineering or chemical synthesis \> The antigens may be used in their native state or may be detoxified or otherwise modified by chemical or physical means and may be aggregated, polymerised or conjugated to a carrier to increase their immunogenicity \> Vaccines may contain an adjuvant (improves the immune response of a vaccine). Where the antigen is adsorbed on a mineral adjuvant, the vaccine is referred to as ‘adsorbed’.

Question 36

What is the difference between active and passive immunisation?

Answer 36

**Passive** * Administration of specific antibodies can be utilised for shortlived immunological protection of the host **Active** * Referred to as active immunisation, because the host’s immune system is activated to respond to the “infection” through humoral and cellular immune responses, resulting in adaptive immunity against the particular pathogen * **The immune response is generally highly specific**: it discriminates not only between pathogen species, but often also between different strains within one species (e.g., strains of meningococci, poliovirus, influenza virus).

Question 37

Describe what live attenuated vaccines and non-living vaccines are. Give examples of each vaccine.

Answer 37

**Live attenuated vaccines** * a first step to improved live vaccines was the attenuation of virulent microorganisms by serial passage and selection of mutant strains with reduced virulence or toxicity * Examples are vaccine strains for oral polio vaccine, measles-mumps-rubella (MMR) combination vaccine, and tuberculosis vaccine consisting of bacille Calmette-Gue´rin (BCG) **Non-living vaccines (two types)** * Inactivation of whole bacteria or viruses \> A number of reagents (e.g., formaldehyde, glutaraldehyde) and heat are commonly used for inactivation * Subunit vaccines: eg Diphtheria and tetanus toxoids

Question 38

What are the FIVE factors for design criteria for vaccine formulation?

Answer 38

1. Selection of vaccine immunogen \> Select the ‘correct’ immunogens that are likely to induce immune response \> Must be the appropriate target and induce immune responses that are effective against the pathogen 2. **Adjuvants and delivery systems** \> improve immune response to vaccine \> examples: colloidal aluinium salt (hydroxide, phosphate) * A depot effect leading to slow antigen release and prolonged antigen presentation * Attraction and stimulation of APCs (antigen presenting cells) by some local tissue damage and binding to pattern-recognition receptors present on APCs * Delivery of the antigen to regional lymph nodes by improved antigen uptake, transport and presentation by APCs 3. **Immunisation schedule** * Depends on the type, magnitude and duration of the immune response * Initial immunisation and subsequent or booster immunisations 4. **Route of delivery** * Parenteral delivery common * Oral delivery (advantage: lead to the induction of mucosal immunity) 5. **Stability** * Avoid degradation * Liquid formulation vs powder form * Temperature control