Biopharmaceuticals Flashcards
What are the chemical changes that can occur to degrade protein formulations? [8]
- Deamidation especially on Asn and Gln residues.
- Amide bond hydrolysis (especially Asp)
- Elimination of water.
- Disulphide bond reshuffling
- Oxidation (Met and Cys) - some salts and metal ions in processing equipment can exacerbate this issue. The use of anti-oxidants or sacrificial amino acids/protein (HAS) can prevent this.
- Cross-linking
- Thiol-disulphide exchange
- Changes in ionisation due to pH variations - use of appropriate buffers can protect against this.
Which residues are particularly prone to deamidation?
Asn + Gln
Which residue is particularly prone to amide bond hydrolysis?
Asp
Which residues are particularly prone to oxidation?
Met + Cys
The presence of what in a formulation can enhance oxidation?
Some salts and metal ions in processing equipment.
How can oxidation of the formulation be protected against? [2]
- Anti-oxidants
2. Sacrificial amino acids/protein (HAS)
How can changes in formulation ionisation due to pH variations be protected against?
Use of appropriate buffers,
In what ways can protein unfolding/aggregation occur? [4]
- Via agitation.
- Via exposure to interfaces (aq/air, aq/organics in presence of solvent)
- High concentration of proteins.
- Exposure of the internal, hydrophobic residues.
How can we limit the aggregation of protein formulations? [2]
- Avoiding conditions that promote protein aggregation during processing:
Use of sugars/polyols or polymers to induce preferential exclusion of solute molecules and human serum albumin or cyclodextrins to stabilise any unfolding. - Supercritical fluids can be used in processing to minimise interfaces.
How can we induce preferential exclusion of solute molecules to limit protein aggregation?
We can use sugars/polyols or polymers.
What are the four potential strategies that could be used for nucleic acid-based therapy?
Addition of genes.
Inhibition of gene expression.
Immunotherapy
Genome editing.
How can the addition of genes be beneficial in treatment of diseases such as cancer? [6]
- Gene replacement - correction of monogenic diseases.
- Genes to code for therapeutic proteins.
- Genes that code for suicide enzymes. (GDEPT)
- Genes that code for ‘soluble’ receptors.
- Genes that code for mAbs to target molecules involved in cancer.
- Genes that code for tumour suppression genes.
What is GDEPT?
Gene-directed enzyme prodrug therapy.
• Suicide gene inserted/targeted for insertion into tumour cells.
• Gene codes for an enzyme that when expressed catalyses/activates a prodrug which has been administered to the body. This prodrug can itself be targeted only to tumour cells via aptamers, affibodies etc. or it can be present homogeneously throughout the body.
• This prodrug is converted into an active form that leads to tumour cell death.
Genes that code for soluble receptors can be used to inhibit tumour progression. What is one molecule that may be targeted in this fashion?
VEGF.
Mucagen - the first aptamer and RNA drug treats ‘wet’ age-related macular degeneration by binding to VEGF and reducing blood vessel growth.
What is Mucagen?
Mucagen - the first aptamer and RNA drug treats ‘wet’ age-related macular degeneration by binding to VEGF and reducing blood vessel growth.
It does however have to be delivered every 6 weeks via injection to the eyes.
How can inhibition of gene expression be accomplished?
- Anti-sense and decoy nucleotides.
2. RNA interference.
What is decoy oligonucleotide therapy?
Use of decoy oligonucleotide sections to interfere with the transcription of genes coding for pro-tumour/illness proteins.
What is anti-sense oligonucleotide therapy?
Use of anti-sense oligonucleotides to interfere with the translation of disease causing proteins.
What is the difference between anti-sense and decoy oligonucleotide therapy?
Anti-sense interferes with mRNA translation in the cytoplasm.
Decoy interferes with mRNA transcription in the nucleus.
How can RNA interference lead to inhibition of gene expression?
siRNA – if the sequence of a disease causing gene is known, we can use small interfering RNA (siRNA) to ‘knock down’ gene expression.
miRNA – MicroRNA is derived from genes that specifically code for miRNAs not proteins. Important in gene regulation. DNA coding for under expressed or missing miRNAs could be delivered to cells to replace the missing miRNAs.
What is siRNA used for?
RNA interference, if the sequence of a disease causing gene is known, we can use small interfering RNA (siRNA) to ‘knockdown’ gene expression.
What is miRNA?
MicroRNA derived from genes that code for miRNA not proteins. Important roles in gene regulation. DNA that codes for missing or underexpressed miRNA could be delivered to cells.
How can immunotherapy be used to treat cancer?
- A tumour biopsy is performed and tumour specific RNA extracted as cDNA before amplification to create a vial of amplified tumour specific RNA.
- Leukapheresis of a patient’s blood takes place to extract mononuclear cells. These are then stimulated to differentiate into monocytes and then into immature Cytotoxic T lymphocytes.
- Tumour RNA loading/exposure to the patient’s immature CTL occurs and the resulting mixture of activated CTLs is administered to the patient.
This is a means of activating a patient’s immune system to fight cancer cells.
`Where must genes, decoy oligonucleotides and genome editing be targeted to?
The nucleus
Where must anti-sense RNA, siRNA and miRNA be targeted to?
The cytoplasm.
What is the most common way of delivering genes?
66% viral delivery. Viruses have evolved to invade cells and deliver genetic material to the nucleus. The viral genes involved with replication are removed (non-pathogenic) and replaced with therapeutic genes.
