Sterile Pharmaceuticals and Pharma-Biotech Products II

Question 1

What is downstream process?

Answer 1

Downstream process is the stage of pharm-biotech production that is concerned with the isolation and purification of targeted compounds synthesised by the host cells.

Question 2

Mention 3 techniques employed in separation and purification of pharm-biotech products.

Answer 2

1. Size exclusion chromatography (Gel filtration)
2. Ion exchange chromatography
3. Affinity chromatography.

Question 2

Briefly describe ion exchange chromatography.

Answer 2

It is a chromatographic technique technique utilised in the purification of pharm-biotech products, which relies on the charge on the molecule of interest as a basis for separation.

The stationary phase consists of resins that are covalently linked to negatively or positively-charged groups.

When the mixture is passed through the column, the molecules of with the opposite charge of the resins bind to the resins and are retained, while the unwanted molecules elute first.
The molecules of interest are then unbound from the resin using the proper salt concentration.

Question 3

Briefly describe affinity chromatography.

Answer 3

This is a chromatographic technique that relies on the affinity of the molecule of interest (e.g. protein) to a known ligand.
The ligand is bound covalently to a resin.

When the mixture is passed through the column, the protein of interest binds to the ligand, while the rest of the mixture elutes first.

The protein of interest is then unbound from the ligand using various methods, e.g. by using competitive molecules or by changing the pH to disfavour the binding of the molecule to the ligand.

Question 4

What is the characterisation of biotech-based product all about?

Answer 4

It is carrying out extensive analysis that involves various methods to characterise the physicochemical and biological properties of the produced compound.

Question 5

Mention 3 techniques involved in the characterisation of biotech-based product.

Answer 5

Spectroscopic methods such as:
1. UV spectroscopy
2. Fourier transform infrared spectroscopy
3. Fluorescence spectroscopy

4. Gel electrophoresis e.g. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Biological assays such as
5. Ligand binding assays
6. Cell-based assays
7. Whole animal assays

Question 6

What are ligand binding assays used for?

Answer 6

They are used for testing the binding of the molecules, such as proteins, to the ligand. This is done in vitro.

An example is the enzyme-linked immunosorbent method

Question 7

Why are pharma-biotech products more challenging in terms of formulation stability?

Answer 7

Because the products are biomolecules such as proteins, peptides and nucleic acids, which have a relatively large molecular size and are held in their correct 3D structures via noncovalent structures, making them more susceptible to chemical and physical degradation.

Question 8

Mention 5 different pathways of chemical biodegradation in pharma-biotech products.

Answer 8

1. Deamidation reaction
2. Racemization reaction
3. Hydrolysis
4. beta-elimination
5. Oxidation
6. Formation of disulfide bonds.

Question 9

Briefly describe the deamidation reaction and provide an example.

Answer 9

In this reaction, the amide side of an asparagine or glutamine residue is hydrolysed to the corresponding carboxylic acid.

Example: the adrenocorticotropic hormone, which is a peptide hormone, can suffer deamidation.

Question 10

Briefly describe the racemization reaction and provide an example.

Answer 10

This involves the inversion of the chiral centre in amino acid residues. This reaction proceeds in basic media.

Example: Casein can exhibit racemization at various amino acid residues.

Question 11

Give an example of a pharma-biotech product that may undergo chemical degradation via hydrolysis.

Answer 11

Secretin can suffer from hydrolysis of aspartic acid in acidic media.

Question 12

Mention 5 factors that may influence the physical degradation of pharma-biotech products.

Answer 12

1. Heat
2. pH
3. Formulation excipients: the presence of other molecules that may disrupt the native structure of the macromolecules.
4. Ionic strength
5. Agitation

Question 13

What is denaturation? What are the types?

Answer 13

Denaturation is the unfolding of protein and the distortion of the secondary and tertiary structure, leading to loss of activity.

The types are:
1. Reversible denaturation: the secondary and tertiary structure of the protein can be repaired by removing the causative agent.
2 Irreversible denaturation: the correct secondary and tertiary structure of the protein cannot be regained by removing the causative agent.

Question 14

What are two instability problems that may occur as a result of denaturation?

Answer 14

1. Aggregation
2. Precipitation

Question 15

Briefly discuss 5 strategies employed to aid the stability of pharma-biotech products.

Answer 15

1. Addition of surfactants: This prevents the adsorption of low-dose protein, such as interferons on the walls of the container. It also prevents aggregation under mechanical stress such as shaking and liquid-container contact.
2. Addition of antioxidants or chelators: Antioxidants, such as methionine and chelators, such as EDTA are used to prevent oxidation, fragmentation and heavy metal-induced complications.
3. Use of Teflon coating on the containers: This also prevents the adsorption of low dose-proteins to container walls.
4. Use of high concentrations of sugars, sugar alcohols and certain amino acids in protein solutions: This provides isotonicity without increasing ionic strength and provides thermodynamic stabilization of protein solutions through preferential exclusion.
5. Dry formulations: This ensures stability through the elimination of water molecules using techniques such as freeze drying and spray drying.
6. Nitrogen gassing to remove oxygen

Spray drying is still a niche process used only in the context of pulmonary delivery of proteins.

Question 16

What are the two theories for the mechanism behind the prevention of aggregation under mechanical stress using surfactants?

Answer 16

1. The replacement of the protein from the surfaces, thereby reducing its exposure to unfavourable void spaces
2. The surfactant covers more hydrophobic patches on the protein, preventing them from interacting and reducing the probability of aggregation.

Question 17

Why is citrate, despite being an effective chelator and a standard buffer excipient, not ideal for formulations?

Answer 17

It has reported to have a slight burning effect at the sight of injection in subcutaneous use and is therefore patient-unfriendly.

Question 17

What is the use of the amino acid, arginine in protein solution formulations?

Answer 17

1. It is a solubiliser for protein drugs such as tPA.
2. It improves the solubility of monoclonal antibodies due to its chaotropic effect.