Sterile Pharmaceuticals and Pharma-Biotech Products I

Question 1

What was the first approved recombinant drug and when was it approved?

Answer 1

Recombinant insulin.
1982

Question 2

Define Recombinant Products.

Answer 2

Recombinant products are products of rDNA technology, which are produced by genetic modification in which DNA coding for a required product is introduced, either by means of plasmid or virus, into a suitable microorganism or cell line in which the DNA is expressed and translated into protein which is then extracted and purified for use.

Question 3

Recombinant products are always________.

Answer 3

Proteins or peptides

Question 4

On what basis are recombinant drugs defined?

Answer 4

Their concrete production process.

Question 5

What are the classifications of recombinant drugs?

Answer 5

i. Class I
ii. Class II
iii. Class III
iv. Class IV

Question 6

What are Class I recombinant drugs?

Answer 6

Class I recombinant drugs differ from the authentic biomolecules due to technological limitations. They are the early approved molecules which had to be produced in E. coli for the lack of other expression systems.

Question 7

What are the typical deviations of Class I recombinant drugs?

Answer 7

i. Substitution of cysteine with serine to prevent the formation of improper disulfide bonds.
ii. An additional methionine at the protein’s N-terminus
iii. The complete absence of glycosylation structures.

Question 8

What are Class II recombinant drugs?

Answer 8

They are more or less authentic copies of biomolecules and correspond exactly to their prototypes in regard to amino acid sequence.

They might however differ in regard to post transitionally added modifications.

Question 9

What are Class III recombinant drugs?

Answer 9

They contain structural variations which are deliberately introduced, mainly to improve pharmacokinetic properties and in some cases, pharmacodynamics.
Example: Pegvisomant (a human growth receptor antagonist resulting from the modification of several amino acids in the human Growth Hormone)

Question 10

What are Class IV recombinant drugs?

Answer 10

They are newly invented, artificial proteins.

Question 11

Mention 5 recombinant antidiabetic drug products, their generic names and expression systems.

Answer 11

i. Insuman® Rapid - Human Insulin, E. coli
ii. Insuman® Basal - Human Insulin, E. coli
iii. Insuman® Comb 15 - Human Insulin, E. coli
iv. Insuman® Comb 25 - Human Insulin, E. coli
v. NovoRapid® -insulin Aspart, S. cerevisiae

Question 12

Mention 5 recombinant antirheumatic/anti-inflammatory drugs products, their generic names and expression systems.

Answer 12

i. MabThera® - Rituximab, CHO
ii. Enbrel® - Etanercept, CHO
iii. Arcalyst®- Rilonacept, CHO
iv. Remicade®- Infliximab, Murine cell line
v. Kineret®- Anakinra, E. coli

Question 13

Mention 5 recombinant Tumor drugs, their generic names and expression systems.

Answer 13

i. MabThera® - Rituximab, CHO
ii. Proleukin® - Aldesleukin, E. coli
iii. Referon®-A - Interferon alfa-2a, E. coli
iv. Intron A® - Interferon alfa-2b, E. coli
v. Neupogen® - Filgrastim, E. coli

Question 14

Mention 5 recombinant anti-anemic drugs, their generic names and expression systems.

Answer 14

i. Erypo® - Epoetin alfa, CHO
ii. Eprex® - Epoetin alfa, CHO
iii. Binocrit® - Epoetin alfa, CHO
iv. NeoRecormon® - Epoetin beta, CHO
v. Dynepo® - Epoetin delta, Human cell line

Question 15

Mention 5 recombinant vaccines, their generic names and expression systems.

Answer 15

i. HBVAPRO® - Hepatitis B vaccine, S. cerevisiae
ii. Fendrix® - Hepatitis B vaccine, S. cerevisiae
iii. Cervarix® - HPV vaccine, Baculovirus expression system
iv. Gardasil® - HPV vaccine, S. cerevisiae
v. Silgard® - HPV vaccine, S. cerevisiae

Question 16

The production of biopharmaceuticals is strictly regulated and based on the directions given in _____________.

Answer 16

The European Pharmacopoeia.

Question 17

What are the steps involved in the formulation of sterile biopharmaceuticals?

Answer 17

i. Development of a host-vector system and its detailed characterization,
ii. Production of a validated master cell bank and derived working cell banks,
iii. Fermentation, protein isolation and purification.
iv. Extensive analytical testing and evaluation of the protein product,
v. Detailed documentation of every single step.

Question 18

The production of biotechnology-based products is divided into two main stages, which are:

Answer 18

1. Upstream processing
2. Downstream processing

Question 19

What is upstream processing?

Answer 19

This is the stage where the targeted compound is synthesised and increased quantitatively by the host cells.

Question 20

What is downstream processing?

Answer 20

This stage is concerned with the isolation and purification of the targeted compounds synthesised by the host cells.

Question 21

Summarise the steps involved in the upstream process.

Answer 21

1. Isolate the gene that codes for the targeted compound.
2. Clone the gene.
3. Insert the gene into a suitable vector molecule e.g. plasmid. [This facilitates the replication and expression of the gene in the host cell]
4. Insert vector molecule containing gene of interest into the host cell, which will express the gene and produce the target molecule.

Question 22

What is gene cloning?

Answer 22

It refers to the isolation and replication a fragment of DNA without alteration in the nucleotide sequence.

Question 23

What is recombinant DNA technology?

Answer 23

Recombinant DNA technology involves the isolation, cloning and insertion of a gene by proper methods into a host cell which then expresses the gene as a part of its genome to produce the protein or peptide of that gene.

Question 24

What is a plasmid?

Answer 24

A plasmid is a small circular DNA molecule that exists and replicates independently from the chromosomal DNA within the bacterial cell.

Question 25

Mention 4 requirements for a vector?

Answer 25

1. It must be well-characterised with known restriction sites that permit the use of restriction enzymes to cleave it to insert the gene of interest and rejoin it with proper ligase enzymes.
2. It must have an origin of replication to be able to replicate within the host cell.
3. A promoter sequence must be present in the vector to ensure efficient transcription.
4. A method of differentiating and separating cells that take up the vector from cells that don’t.

Question 26

How are cells that take up the plasmid vector differentiated and separated from cells that don’t?

Answer 26

An antibiotic- resistance gene, such as streptomycin resistance gene is usually inserted in the vector. The cells are then cultured in a medium that contains the antibiotic. The cells that don’t take up the vector die, leaving the cells that do.

Question 27

Mention one method of inserting plasmid vector into the host bacterial cell.

Answer 27

Electroporation.
It involves the use of electromagnetic energy to create pores in the cell walls of the bacterial cells through which the plasmid vectors can enter.

Question 28

The choice of host cell depends on several factors, which include:

Answer 28

1. Economic factors
2. The presence or absence of post-translational modifications.

Question 29

What are the advantages and disadvantages of bacteria host cells such as E. coli?

Answer 29

Advantages:
i. They are well-understood for the purpose
ii. They are cheap to culture and grow

Disadvantage:
i. Their inability to carry out post-translational modification on the synthesised protein or peptide e.g. glycosylation.

Question 30

Mention 3 host cells that can carry out post-translational modification.

Answer 30

Eukaryotic cells such as:
i. Yeast cells
ii. Chinese Hamster Ovary (CHO) cells
iii. Baby Hamster Kidney cells
iv. Mammalian cells.

Question 31

What are the advantages and disadvantages of using mammalian cells for gene expression?

Answer 31

Advantage:
i. They can carry out proper post-translational modifications as well as correct folding of therapeutic proteins.

Disadvantage:
They produce relatively low yield.

Question 32

Define DNA library and give 2 types.

Answer 32

A DNA library is a collection of DNA fragments that have been cloned and inserted into host cells for storage.

The two types are:
i. Genomic DNA libraries
ii. Complementary DNA (cDNA) libraries

Question 33

Briefly describe Genomic DNA libraries and Complementary DNA libraries.

Answer 33

Genomic DNA libraries are created by treating the entire DNA of an organism to cleave the DNA and generate smaller DNA fragments which are then inserted into vectors, and then host cells.

Complementary DNA libraries are constructed from the mRNA molecules present in a tissue, which are then converted into complementary DNA (cDNA) by the action of reverse transcriptase enzyme.