biologics

Question 1

what are biomacromolecular drugs

Answer 1

large (MW>2kD) biological molecules with therapeutic affect that are not accessible by chemical synthesis

Question 2

what are the key differences between small molecule drugs and biologics

Answer 2

biologics cannot be chemically synthesisedbiologics are not membrane permeable biologics are more sensitive to storage/handling

Question 3

give an example of a biomacromolecule

Answer 3

insulin

Question 4

how is insulin produced

Answer 4

recombinant DNA technology

Question 5

what are the steps in recombinant DNA technology

Answer 5

1. gene identification, amplification, isolation 2. gene integration to vector 3. vector into host cell 4. cell growth5. cells containing target identified 6. isolation/purification

Question 6

what are the options for hosting the biologics during amplification

Answer 6

e coli yeast animal cells insect cells plant cells

Question 7

what are the advantages to using e coli as a host cell

Answer 7

well characterised biology high expression of heterologous proteins quick and cheapcan be scaled up

Question 8

what are the disadvantages to using e coli as a host cell

Answer 8

heterologous proteins accumulate intracellularlyno PTMpresence of pyrogenic LPS formation if inclusion bodies

Question 9

what does Heterologous mean

Answer 9

protein of interest does not occur naturally in the cell

Question 10

what are inclusion bodies

Answer 10

insoluble aggregates of partially soluble heterologous proteins

Question 11

what are PTM

Answer 11

Any covalent modification of peptide sequence once it has been formed

Question 12

which cell types can perform PTM

Answer 12

Prokaryotes do not have the necessary glycosylation machinery, so using eukaryotes to produce the protein (Chinese hamster ovary cells)

Question 13

what are two examples of PTM

Answer 13

glycosylation, phosphorylation

Question 14

what are the two types of glycosylation

Answer 14

n-linked o-linked

Question 15

what happens in n-linked glycosylation

Answer 15

Transfer of 14-mer oligosaccharide donor to protein as it emerges from rough ER - catalysed by oligosaccharyl transferase

Question 16

what happens in o-linked glycosylation

Answer 16

involves serine/threonine residues

Question 17

how does glycosylation work

Answer 17

requires sugar donor and an acceptor

Question 18

what is a glycoform

Answer 18

Variations in glycosylation patterns, have different stability, solubility, half life, biological activity and immunogenicity

Question 19

how are monoclonal antibodies manufactured

Answer 19

hybridoma technology

Question 20

how does hybridoma technology work

Answer 20

○ Mouse immunised and produce plasma cells/ antigens ○ Plasma cells mixed with immortal myeloma and fused with PEG ○ Cells grown in HAT medium - only allows fused cells to grow ○ Fused cells showing high desired protein are expanded and grown in large numbers

Question 21

how is the desired protein recovered from host cells

Answer 21

1 from cell 2 chromatography

Question 22

how is the desired protein recovered from host cells - cellular

Answer 22

1) Chemical treatment 2) Sonication/homogenisation 3) Agitation in presence of abrasives

Question 23

how is the desired protein recovered from host cells - chromatography

Answer 23

1) Affinity chromatography 2) Ion exchange chromatography 3) Gel filtration chromatography

Question 24

what are biosimilars

Answer 24

biologics cannot be copied due to complexity of processing, Any biologic that results from this repeat process is called a biosimilar