Biopharmaceuticals Flashcards
What are pharmaceuticals?
Drugs used to diagnose, prevent, treat or cure disease
What is a biotechnology medicine/product?
Pharmaceutical product used for therapeutic or in vivo diagnostics, produced in full or in part by modern biotechnological means (include antibiotics extracted from fungi; insulin from pig pancreas or recombinant insulin)
Give examples of products which are from a animal origin
Antibodies, enzymes, hormones, blood factors, growth factors, cytokines
Give examples of products which are form a plant origin
aspirin, alkaloids, digitalis, quinine
Give example of products form a microbial origin
vaccines, antibiotics, toxins/toxoids
What are biopharmaceutical proteins or nucleic acids?
based pharmaceuticals that are used for therapeutic or in vivo diagnostics, produced by means other than direct extraction from native biological source
Give examples of pharmaceuticals from chemical synthesis?
Paracetamol (acetaminophen), ketamine, acyclovir
Give examples of pharmaceuticals from biological extraction
Factor VIII, insulin, alkaloids (morphine), antibiotics, vaccines, antibodies (passive immunity), thrombolytic agents
Give examples of some biopharmaceuticals
Factor VIII, insulin, vaccines, antibodies (e.g. monoclonal), thrombolytic agents
What are the advantages of biopharmaceuticals?
Overcomes issue of source availability (e.g. Cytokines)
Improves product safety (e.g. Blood products – HIV, hepatitis, CJD)
Alternative to extraction (e.g. Insulin from slaughterhouses, hormones from urine)
Engineering of more effective therapeutic proteins (e.g. Insulin, Factor VIII)
What region of a chromosome is the encoding region?
euchromatic gene
What fraction of the genome is transcribed into RNA?
1/3
What % of RNA is believed to produce proteins?
~5%
What are bioinformatics used for?
to identify functional sequence motifs (e.g. catalytic sites, transmembrane regions) by homology to other known genes (homologues), even in different species (orthologues)
What are knock-out animals?
generation of animals in which the study gene is deleted – observation of phenotype can Often reveal role of gene product
What are the different variations in gene expression between cells?
different cell types - stage in organisms development - phase of cell cycle - response to internal and external (i.e. environmental) factors
What are gene chips?
study of differential RNA expression (transcriptome) in different cells/conditions (e.g. Expression in cancer cell may provide drug target)
What are proteomics?
Proteomics: analysis of cellular protein expression (proteome)
What are structural genomics?
3D protein analysis (NMR) – structure may predict function
What is pharmacogenomics?
correlation of gene sequences to drug response
Determine which drugs (& dose) to give which individuals – benefits v side effects
What do pharmacogenomics look at?
mutations and polymorhisms (single nucleotide polymorphisms – SNPs)
what are most BPs produced by?
genetic engineering using recombinant expression systems
What are the steps of using recombinant expression systems?
First - identification and isolation of therapeutic gene/custom synthesis
Molecular cloning into vectors (e.g. plasmids, viruses)
Propagation (maybe protein expression) in cell lines
Purifying products (DNA, protein, vector)
What is used to clone mRNA into cDNA?
produced using retrovirus reverse transcriptase
What regulates the expression of genes?
Gene promoters
What does differential splicing allow?
Different mRNAs and thus proteins to be formed from the same gene sequence
What are introns?
Non coding DNA
What is the gene often fused to in order to ease purification?
A leader sequence or tag that allows the protein to be isolated using, for example antibodies
What are the two types of expression a cell line can have?
Transient/stable expression
What cell lines are most BP proteins produced in?
E.coli (e.g. K12) or mammalian cell lines (e.g. Chinese hamster ovary, CHO; baby hamster kidney, BHK)
What does poor post-translational modifications affect?
Functions
What is removed form microbial cell lines?
Surface LPS pyrogenic
What can high-level intracellular expression of heterologous proteins produce?
Inclusion bodies
What is a advantage of inclusion bodies?
Can aid centrifugal privation
What is a disadvantage of inclusion bodies?
Aggregates of partially folded foreign proteins
What is a solution of inclusion bodies?
express soluble intracellular fusion protein/tag (foreign-native protein)
Purify and enzyme cleave off target protein
What are some examples of fusion proteins?
Fusion proteins include glutathione transferase, thioredoxin, maltose-binding protein
What are advantages of culturing yeast cell line?
Cultured relatively fast and cheaply
What is the most widely used yeast cell line?
S. cerevisiae most widely used (e.g. insulin, Hepatitis B vaccine antigen, anticoagulants)
What are advantages of using S. cerevisiae?
Genetics well characterised
Safe – used in brewing, baking
What does Mannose glycosylation lead to?
rapid blood clearance
What are disadvantages of mammalian cell lines?
Slower and more expensive to set-up, up-scale and maintain (nutrients, fragility)
What are most widely used mammalian cell lines?
Chinese Hamster Ovary (CHO), Baby Hamster Kidney (BHK) most widely used (e.g. insulin, vaccines). Hybridoma cells for monoclonal antibodies
How are transgenic animals produced?
by microinjecting exogenous DNA construct into egg cells
What are some advantages of transgenic animals?
Milk safe, well characterised, abundant
Continued inheritance of recombinant DNA in every cell
Relatively low costs after initial transgenic outlay
What is a disadvantage of transgenic animals?
significant lag period from viable embryo and manufacture (n.b retrovirus vectors)
What is the only licensed transgenic animal BP?
human antithrombin ‘Atryn’
Give a example of some glycosylation differences using transgenic animals?
c.f. CHO cells – seemed to enhance efficacy but reduce half-life
What do Agrobacteria species contain?
Ti plasmids
What are advantages of transgenic plants?
Cultivation/harvest/scale up easy and costs low
Proteins usually stable
Plants free of human pathogens
Can get low expression/gene silencing
What are disadvantages of transgenic plants?
Glycosylation significantly different – immunogenic
Potential plant metabolite contamination (alkaloids)
Public issues surrounding GM crops – cell lines?
What is CaroRX?
recombinant ‘plantibody’ against Strepococcus mutans in tooth decay - topical BP
What is Merispace?
oral (corn) recombinant gastric lipase to treat lipid malabsorption due to poor pancreas secretion (e.g. CF)
What can Agrobacteria and plant viruses also be used for?
Transferring BP genes into plants transiently
What are advantages of insect cells?
High level expression (strong promoters) – up to 50% of cell protein
Rapid and cheaper culture than mammalian cells
Most human pathogens don’t infect insect cells
What are disadvantages of insect cells?
Most protein is intracellular – purification needed
Often incomplete/markedly different glycosylation
Cervarix HPV cervical cancer vaccine particles from?
recombinant L1 capsid protein
