industrial production

to be active and function proteins must

have correct AA sequence

have correct folding

no aggregation

have correct PTM

phosphorylation

phosphate on serine theonine or tyrosine (OH)

glycosylation

sugar to N of asparagine

O of serine or theonine

ubiquination

add to lysine for degregation

sumoylation

adds SUMO protein

acetylation

adds acetyl to n-terminal at lysine

lipidation

adds lipid

methylation

adds methyl to lysine or arginine

hydroxylation

adds hydroxyl

what percent of marketed biopharm are glycosylated

70%

what is the con of natural sources of proteins

expensive

serum sickness or risk of prion disease

steps for protein production

find gene sequence for protein

obtain best source for nucleic acid

clone into vector

add vector into cell and ferment

what are the sources of finding the gene

gene synthesis

genomic DNA

cDNA

gene synthesis

preferred choice but you must know the exact sequence

gDNA

contains all protein information but small amount of protein of interest

cDNA

extract mRNA of gene and convert back into DNA

how to modify and optimize proteins

must alter coding nucleic acid with site directed mutagenesis

what are the components of a vector

MCS

promoter

antibiotic resistance market

origin or replication

where is the recominant dna inserted into a vector

MCS

prokaryotic living system

no PTM

protein may aggregate

yeast living system

some PTM but does not do all

mammalian cells living system

good for large proteins and has all PTM

which biopharm are produced in E coli

neulasta (pegulated filgrastum)

roferon-a (INF a2a)

which biopharm are produced in Vibrio cholera

dukoral

cholera toxin subunit B

which biopharm is produced in a fungi

Gardasil

what is the cons of using fungi

high cost of purification and difficult to cultivate

what biopharm are produced in CHO

humira

enbrel

what biopharm is produced in plants (nicotiana benthamiana)

3 humanized mAB for Ebola

what selections are required for fermentation

cell line

culture media

growth parameters

process optimization

what is a master cell bank

pool of cells from a single clone that is split into aliquots and stored

serves as the primary stock of cells for production

what is the working cell bank

derived from master cell bank that used for production of biopharm