Industrial Production

physiological protein production

• DNA unwinds from histones → RNA polymerase attach to start of gene

• transcribe mRNA using complementary bases → mRNA to ribosome to be translated → amino acid chains

for activity/functionality, proteins must:

• correct AA sequence

• folded correctly (tertiary and quaternary)

• not aggregate/precipitate

• correct post-translational modification

post translational modification

• are — changes to —

• occur in —

• covalent changes to R-group, peptide bonds, or N/C terminals

• occur in multiple organelles

types of post-translational modification (PTM)

• phsophorylation

• glycosylation

• ubiquitination

• sumosylation

• oxidation (disulfide bond)

• acetylation

• lipidation

• methylation

• hydroxylation

phosphorylation

add phospate to serine, threonine, tyrosine (AAs containing OH)

glycosylation

• attaches to sugar to either an N or O atom in AA side chain

• usually on asparagine or O of serine/threonine

• N-linked oligosaccharides all have pentasaccharide core

  • pentasaccharide core = 3 mannose sugars + 2 N-acetylglucosamine

ubiquitination

adds ubuquitin to lysine residue to induce degradation

sumosylation

adds small protein sumo (small ubiquitin-like modifier) to target protein

oxidation (disulfide bond)

• covalently binds to sulfur atoms of 2 different cysteine residues

  • thiol groups (SH) go thru this process to form disulfide bond

acetylation

adds acetyl group (CH3CO) to N-terminal of protein or lysine residue

lipidation

adds lipid to protein chain

methylation

adds methyl (CH3) usually to lysine or arginine residue

hydroxylation

adds OH group to side chain

natural sources of proteins

• nonhuman animals or bacterial proteins

  • expensive or cause serum sickness

  • risk of prion (neurodegenerative disease) form misdfolded proteins

natural sources of protein examples

• insulin

  • from bovine/porcine pancreas

  • botox

    • from botulinum toxin A of Clostridium botulinum

  • collagenase

    • from Clostridium botulinum

  • diphtheria antitoxin

    • from serum of immunized horse/sheep

  • venom antidotes

production line

upstream → downstream → formulation → packaging

• optimization occurs at every step

protein production steps

• determine gene sequence (nucleic acids) that make desired protein

• obtain best source for nucleic acid

• clone gene into vector

• introduce vector into proper living cells and fermentation

determine gene sequence (nucleic acids) that make desired protein

• use NCBI

• optimization of protein requires modif/engineering → done by nucleic acids

  • Use mutagenesis to insert/delete/sub nucleic acid base pairs that encode key AA residues

obtain best source for nucleic acid

• gene synthesis

• genomic DNA

• mRNA to cDNA (complementary)

1. Gene synthesis 

   1. preferred because it’s less expensive and laborious, but requires knowledge of exact sequence

2. Genomic DNA

   1. contains all DNA info for all proteins, but have less of it for use

3. mRNA to cDNA (complementary)

   1. extract mRNA from organ that expresses it a lot and form cDNA with reverse transcriptase

   2. has desired nucleic acid in large quantity, but is less stable than DNA

clone gene into vector (usually plasmid)

• multiple cloning site (MCS)

• promoter

• antibiotic resistant marker

• origin of replication

1. Multiple cloning site (MCS) → once within a plasmid to allow DNA insertion into region

2. Promoter → DNA sequence for polymerase to bind and initiate transcription

3. Antibiotic Resistant Marker → added so that produced protein is resistant to a specific antibiotic

   1. Allows screening since cells that didn’t intake DNA for resistance and replication will be removed by antibiotic

4. Origin of replication → where DNA replication is initiated in DNA sequence (not transcription)

Introduce vector into proper living cells and fermentation

Types of living cells: prokaryotes (E.coli), yeast, mammalian cells (CHO, HEK293, insect)

prokaryotes

• Easy manipulation and rapid growth

• Large scale fermentation

• Simple, low-cost

• High yield

• Good for SMALL PROTEINS (<30,000 Da)

• Production within the cells

• Almost no PTM (post-translational modification)

• Aggregation possible ← proteins may not fold correctly

• Not good for large proteins since folding may be difficult

yeast

• Rapid growth

• Large scale fermentation

• Some PTM performed

• Some protein production within the cells

• Does not do ALL PTMs

• May do PTM differently

mammalian/insect

• Good for LARGE PROTEINS

• Performs ALL PTMs

• Proteins are secreted into media

• **Note: large is >30,000 Da

• [EX.] Humira (adalimumab), Enbrel (etanercept)

• Grows slowly

• Expensive

• Difficult to do large scale

• Requires more technical skill 

bacteria

• e coli (main), vibrio cholera (Dukoral, cholera toxin subunit B), bordetella pertussis

• Nuelasta

  • pegylated Filgrastim (G-CSF)

• Roferon-A

  • interferon a2a

Fungi

• saccharomyces cerevisiae

• difficult to cultivate, expensive to purify

• Gardasil = HPV vaccine

plant based lines

• glycoprotein production in plant systems

• can cause issues due to formation of products that are hyperglycosylated

  • contains sugars that are immunogenic in humans and deviod of sialic acid which will influence serum half life

• ZMapp

  • biopharm: 3 humanized mABs

  • cell line: low nicotine tobacco variety

  • use: ebola product

transgenic animal production systems

express recombinant products in milk (ex: rabbits, goats)

cell growth curve

• protein production is OPTIMAl in LOG (exponential phase) = Most growth

• cells harvested in stationary phase to maximize growth and minimize cell death

fermentation

• requires control of conditions for cell growth and production

• select cell line, culture media, growth parameters + optimize process

what provides control of conditions/environment for recombinant proteins?

bioreactors

bioreactors

• contains everything after upstream process (culture + media)

• maintain temp, pH, gas, etc

cells banks

• master cell bank

• working cell bank

master cell bank

• produced from OG therapeutic producing cell line

• under well-defined conditions

• in ultra-temp (-80C) and vapor phased liquid nitrogen

• pool of cells derived from single clone

if master bank is lost/destroyed, all validation for product must be —

repeated

working cell banks

• derived from master cell bank

• used in production of final biopharm product

• can be obtained from master cell bank if lost