Lecture 3: Industrial Production

To be active and functional, proteins need to what?

• have the correct amino acid sequence

• folded properly (correct tertiary and quaternary structures)

• not aggregated or precipitated

• have the correct posttranslational modifications (PTMs)

  • PTMs are covalent modifications that occur on amino acid side chains, peptide linkages, or at the protein's N- or C- termini

  • PTMs occur in various cellular organelle

What are the types of post-translational modification?

• Phosphorylation

  • Adds phosphate to serine, theonine, or tyrosine

• Glycosylation

  • Attaches sugar usually to “N” of an asparagine or “O” of serine or threonine in AA side chain

• Ubiquitination

  • Adds ubiquitin to lysine residue of target protein for degradation

• SUMOylation

  • Adds small protein SUMO (small ubiquitin-like modifier) to a target protein

• Disulfide bond

  • Oxidation; covalently links the “S” forms of 2 different cysteine residues

• Acetylation

  • Adds acetyl group to an N-terminus of a protein or at lysine residues

• Lipidation

  • Attaches lipid, like a fatty acid to a protein chain

• Methylation

  • Adds methyl group at lysine or arginine

• Hydroxylation

  • Attaches OH (hydroxyl) to a side chain of a protein

How much biopharmaceutical are glycosylated?

• 70% of marked biopharmaceuticals

Where does post-translational glycosylation occur?

• Occurs at the N of an asparagine (Asn-X-Ser or Asn-X-Thr) or O of a Ser or Thr

Glycosylation affects what? It differs between what?

• biopharmaceutical stability, solubility, bioavailability, in vivo activity,
  pharmacokinetics, and immunogenicity.

• It differs between species, different cell types within a species and batches of in cell culture-produced therapeutic protein

In glycosylation, all N-linked oligosaccharides have what?

• have a common pentasaccharide core composed of 3 mannose units
  and 2 N-acetylglucosamine units

  
  

What is oxidation (Disulfide bond)?

• 2 cysteine residues can come together if oxidized which helps with folding properties

What uses non-human animals or bacterial proteins?

• Insulin (extracts of bovine and porcine pancreas)

• Botox (Botulinum toxin A): Clostridium botulinum

• Collagenase: Clostridium botulinum

• Diphtheria antitoxin: from serum of immunized horse or sheep

• Venom antidotes

What are characteristics of natural sources of proteins? (non-human animals or bacterial proteins)

• Expensive

• Can cause serum sickness (hypersensitivity) and risk of prion diseases (CJD)

What are the steps in biopharmaceutical production?

• Upstream → downstream → formulation (dosage form) → packaging (marketing)

What are the steps of protein production?

• 1. Determine the gene (nucleic acid) sequence (NCBI) that encodes the desired protein

• 2. Obtain the best source for the nucleic acid (message)

  • *gene synthesis

  • *genomic DNA

  • *mRNA and cDNA

• 3. Clone the gene into a vector

• 4. Introduce the cloned vector into the proper living cell and fermentation

How do you obtain the gene encoding the protein of interest? What are the pros and cons?

• I. Gene synthesis

  • Pros: less expensive, less labor, preferred choice

  • Cons: requires you to now the exact sequence ahead of time

• II. Genomic DNA

  • Pros: contains all DNA info for all proteins

  • Cons: present in less amount

• III. cDNA (from RNA)

  • Start by extracting the mRNA from the organ that produces this protein in large amount

  • form cDNA

  • Pros: contains the desired nucleic acid in large amounts

  • Cons: less stable than DNA

How can proteins/peptides be modified/optimized?

• Proteins/peptides can be modified or optimized by adding, removing or replacing one or more of amino acid residues

To engineer the protein, what should you do?

• To engineer the protein, you should modify the coding nucleic acid.

  • Using site directed mutagenesis to insert, delete or substitute nucleic acid base pairs that encode
    key amino acid residues

What are the 4 main parts of a vector?

• MCS

• Promoter

• Antibiotic resistance marker

• Origin of replication

What is a multiple cloning site? What does it allow?

• MCS is a short segment of DNA which contains many restriction site

  • Restriction sites should occur only once within a given plasmid

  • Allows a piece of DNA (of interest) to be inserted into that region

What is a promoter?

• A promoter is a sequence of DNA to which polymerase bind that initiate transcription. It activates transcription and translation of code downstream of it

What is an Antibiotic Resistance Marker (selectable marker)?

• A sequence of DNA (gene) that can produce a protein which causes resistance to a specific antibiotic. It selects against those cells that did not take in the foreign DNA

What is the Origin of Replication?

• Sequence of DNA where replication is initiated

For choosing the proper living system, what can we choose?

• Prokaryotes (E. coli)

• Yeast

• Mammalian Cells (CHO, HEK293, or insect)

What are the pros, cons, and protein production of Prokaryotes (E. coli)?

• Pros:

  • Easy manipulation

  • Rapid growth

  • Large scale fermentation

  • Simple and low cost media

  • High yield

  • Low cost

  • Suitable for small proteins

• Cons:

  • Almost no PTM (post translational modifications)

  • May aggregate or not fold properly

  • Not appropriate for large proteins

• Protein production: INSIDE CELL

What are the pros, cons, and protein production of Yeast?

• Pros:

  • Relatively rapid growth

  • Large scale fermentation

  • Performs some PTMs

• Cons: Does not perform ALL PTMs or performs them differently

• Protein production: sometimes

What are the pros, cons, and protein production of mammalian cells?

• Pros:

  • Appropriate for large proteins

  • Performs all PTMs

• Cons:

  • Slow growth

  • Expensive media

  • Difficult to perform large scale

  • Requires more technical expertise

  • High cost (over 1 mil/kg)

• Protein production: secreted in the media

In recent years, how are biopharmaceuticals changed in terms of percent mammalian vs non-mammalian?

• Mammalian in recent years are triple of non-mammalian cells VS back in the 80s

There is heterogenous expression in bacteria, mainly what?

• E coli

What are other bacteria noted besides E coli?

• Vibrio cholera (Dukoral, cholera toxin subunit B)

What biopharmaceuticals are made in bacteria?

• Neulasta → PEGylated Filgrastim (G-CSF) for neutropenia

• Roferon-A → Interferon-alpha2a for chronic hep C, Kaposi’s sarcoma

In Fungi, which biopharmaceutical was made in this?

• Gardasil → HPV Vaccine

In CHO (mammalian), which biopharmaceuticals are produced in these?

• Humira → Adalimumab, Anti-TNF alpha mAB → for inflammation

• Enbrel → Etanercept, Anti-TNF alpha mAB → for inflammation

Which biopharmaceutical was made in Plant Based Lines?

• ZMapp → 3 humanized mABs

  • Cell line: low nicotine tobacco variety Nicotiana benthamiana

  • Use: Ebola product

When bacteria grows, what does the graph look like?

• Number of cells (log) vs Time

  • Starts with lag phase (getting used to fermentation media)

  • Log (exponential) phase (starts growing because low toxic waste + phase where cells are most active and we want the protein production to be made here)

    • We want to stop fermentation before stationary phase

  • Stationary phase (number of cells growing equals those dying)

  • Death (decline) phase (where waste and toxins accumulate and nutrients in media are less so cells rupture and die)

What does fermentation require?

• Requires well controlled conditions for cell growth and biopharmaceutical production

In fermentation, what do we need to have a careful selection of?

• Cell line

• culture media
  (sugars, fat, amino acids, electrolytes, vitamins, trace minerals, hormones, serum)

• growth parameters

• process optimization

What is the yield of fermentation?

• 10-200 mg/L → 1 g/L (Up to 20 g/L)

What is the Master Cell Bank produced from? How is it created under?

• From the original therapeutic producing cell line

• Created under well defined conditions following a detailed procedure

How is the master cell bank split?

• Split into aliquots and stored in multiple vials in ultralow (below -80C) temp freezers and vapor phased liquid nitrogen (cypropreserved)

In the master cell bank, the pool of cells is derived from what? How must the integrity be? What happens if lost or destroyed?

• Pool of cells is derived from a single clone with specific genetic traits

• The integrity must be protected and always activated

• If lost/destroyed all product validation will have to be repeated

Where is the working cell bank derived from? When is it used?

• Derived from the master cell bank

• Used in the production of the final biopharmaceutical product

If the working cell bank is lost, how can it be obtained?

• If lost can be obtained from the MCB