PF2016 10

• Proteins

  • High molecular weight
  • Complex structures – primary, secondary, tertiary, quartery structure and post translational modifications
  • Structural related activity
  • Low stability
• Challenges

  • Demand for affordability and improved access
  • Complexity of biopharma supply chain and operations
  • New manufacturing technology platforms
  • Quality compliance and regulatory scrutiny
• Biosimilar

  (in the EU) A biological medicinal product that contains a version of the active substance of an already authorised original biological medicinal product (reference medicinal product). A biosimilar demonstrates similarity to the reference medicinal product in terms of quality characteristics, biological activity, safety and efficacy based on a comprehensive comparability
• Biosimilar medicines

  • Follow-on versions of original biological medicines
  • Independently developed after the patent protecting the original product has expired
  • Intended to have the same mechanism of action as the original biological medicines, and are designed to treat the same diseases as the innovator's product
• Generics of chemistry based medicines are identical copies of the original product, based on a strict definition of "sameness"
• Analytical and preclinical tests are not sufficient to demonstrate the sameness of two biological products or that they are identical
• Companies starting up new lines of their own biological medicines at new facilities can experience challenges in consistently reproducing the same manufacturing process – cellular factories
• Small Molecular drugs

  vs Macromolecules

  
• Upstream processing

  Initial fermentation process, which results in the initial generation of product
• Downstream processing


  Actual purification of the product and generation of finished product format (i.e. filling into its final product containers, freeze-drying if a dried product format is required), followed by sealing of the final product containers

  
• Bioprocessing

  Initiated with the modification of a cell or micro-organism to introduce a genetic sequence coding for the desired protein. Cell or micro- organism is conserved and used as the master source for manufacturing. Target protein is produced by growing the recombinant source in a bioreactor. Purification of the protein of interest. API stabilised and formulated for use as a therapeutic drug product
• Quality, Safety and Efficacy


  • Ensure that the resulting product remains biologically active for the intended therapeutic indication. Viable manufacturing process is a key determining factor in the development of a biological medicine
• Conventional expression systems

  Primarily derived from mammalian cells, E.coli, and less extent yeasts, insect cells and transgenic animals. Advantage is regulatory acceptance

  
• E.coli (prokaryotic)


  • Insulin, growth hormone, interferon. Proteins where folding and post translational modifications not required for activity. Bacterial and yeast cells contain a cell wall making them sturdier. Protein usually biosynthesised inside the cell necessitating a cell disruption during harvesting. Requires a simple growth medium containing sugar(s) and amino acids or protein digests. Usually heat sterilisable. The cell populations double on the order of minutes; 3-5 days usually required for usable protein levels at harvest. Wider temp and pH tolerated by microbial cells (e.g., temperature +/- 1oC) in fermentation; pH control with NaOH or HCl
• Mammalian (non-human) cell lines (eukaryotic)

  • Chinese hamster ovary (CHO), Baby hamster kidney (BHK21), Murine myeloma cells (NS0 and Sp2/0). Where folding and post translational modifications (glycosylation) are required for activity. Protein is secreted outside the cell, saving a cell disruption step. Mammalian cells lack cell wall, making them more fragile and destroyed by process shear. Can yield potentially adventitious agents such as viruses and mycoplasma, requiring purification validated for their removal. Requires a more complex and expensive growth medium such as mammalian serum or plasma (which, again risk carrying viruses or mycoplasma). Usually filter sterilised. The cell populations double on the order of a day; 4-5 months often required for usable protein levels. Relatively narrow pH and temperature range (temperature +/- 0.1oC); also acid addition must be gentler, e.g. CO2
• S cerevisiae (yeast) cell lines (eukaryotic)

  • Insulin (and insulin analogs), human serum albumin, hepatitis vaccines and virus like particles, e.g., for vaccination against human papillomavirus. Enables production and proper folding of many human proteins post-translational modifications of the protein. Proteins can be secreted to the extracellular medium. Performs high-mannose type N-glycosylation. This confers a short half-life of the modified protein in vivo, which then can have a reduced efficacy for therapeutic use

  
• Insect cells and plants

  • Produce proteins with complex glycosylation patterns; however, the glycan structures produced are significantly different from those produced in humans. ELELYSO approved by FDA for the treatment of type 1 Gaucher disease. This therapy is produced using genetically modified carrot plant root cells that produce the enzyme with a human compatible glycan profile. Insect cells infected with the viral vector baculovirus (baculovirus-insect cell expression system) can also efficiently express recombinant proteins, and these systems are mostly used for the development of vaccines. Transgenic insects in development
• Transgenic animals

  • ATryn, an anticoagulant protein derived from the milk of transgenic goats. Ruconest, recombinant human C1 inhibitor (rhC1INH) milk of transgenic rabbits
• Human cell lines

  • Cell lines are virally transformed, tumour derived and immortalised for high producer cell lines. Per.C6, HEK293 (Xigris), HT-1080 (Elaprase)
• Master Cell Banks (MCB)

  Derived are usually frozen in either freezers or liquid nitrogen. Organisms that are critical to patented processes or that in themselves are patented must be deposited in an internationally recognised, publicly administered collection, access to them can be restricted
• Purpose of master cell bank

  The product is always produced from identical starting material. The cell bank is certified to be free from contaminating organisms. The cell bank is unchanged by repeated passage in culture
• Working Cell Banks (WCB)

  Derived from the Master Cell Bank. Working cell banks that will laterally derive from this have to be validated at the time of their derivation to assure reproducibility and continuity. Master and Working Cell Banks are stored frozen (-80°C) or in liquid nitrogen (-196°C) to minimise mutational risks
• Cell Bank Control

  Working cell banks are generated by propagating the master cell bank in liquid media and filling pre-determined aliquots in cryoprotective solution into sterile vials for frozen storage. Each individual vial is labelled prior to frozen storage in quarantine and representative samples are removed for validation. Once validated, the vials are released from quarantine by the Quality Unit. Each vial then becomes the unique source for each production batch, thereby assuring total traceability
• Cell Bank Validation

  Verifies identity, purity, stability
• Inoculum to Bioreactor

  Propagating the seed inoculum. Defined quantity of WCB is transferred using aseptic technique into the sterilised media. Inoculations are performed by trained suitably garbed personnel in Class 100 Biosafety cabinets. Flasks are re-sealed and removed and incubated for defined time, temperature and agitation conditions as per license and batch record. An uninoculated control from the same media lot is often incubated in parallel. The used vial is also retained for potential testing in the event of a contamination. The option of inoculating a second inoculum is often taken in case of subsequent plant delays and to facilitate a later plant inoculation. After the defined time this shake flask is used as the inoculum for the seed fermentor

  
• Inoculum to Bioreactor

  • Growth rate, temperature, pH, dissolved oxygen, agitation, foam
• Cell-culture media


  Cell line, cell-culture process parameters, cell-culture media are key driver of bio-production processes. Cell-culture media impacts on cell growth and viability, protein production and protein quality. Cell-culture media provide nutrients to the cell and stimulatory and regulatory effects e.g. metals manganese, copper and iron can serve as co-factors for enzyme activation and play a key role in driving protein titre and protein quality. Sufficient levels of amino acids are required for normal cell growth and function, but also for the production of the correct target protein. Media can contain animal-derived components e.g. serum, but has many inherent disadvantages. Therefore the development of serum free media
• Disadvantages of serum, fetal bovine serum

  • Batch-to-batch variation in composition which leads to inconsistent growth and productivity. A high protein content that hinders product purification. Potential for product contamination from unwanted viruses and mycoplasma that may be present in serum as well as the vectors of bovine spongiform encephalopathy (BSE, or "Mad Cow Disease"). Cost is often prohibitive at $500–1,000 per litre and this can account for up to 95 % of the overall cost of the media. Ethical concerns
• • small molecule drugs: low molecular weight, some structures, synthesised by chemical reactions, easily purified, good stability, conventional formulations, predictable pharmacokinetics, little immune response, large scale manufacturing
• macromolecules: large molecular weight, complex structures, synthesised by biochemical processes, unstable, low oral bioavailability, non-liner pharmacokinetics, immunogenicity, complex scale up
• bioprocessing

  modification of a cell or micro organism to introduce a genetic sequence
• expression systems


  e.coli, mammalian cells, yeast lines, insect cells, transgenic animals, human cel lines
• purpose of master cell bank

  identical starting material, free from contaminating organisms, unchanged by repeated passage in culture
• how are working cell banks generated

  propagating the master cell bank in liquid media and filling pre-determined aliquots in cry-protective solution into sterile vials for frozen storage
• cell bank validation

  identity, purity, stability
• areas where culture must be controlled

  1.growth rate 2.temperature 3.ph 4.dissolved oxygen 5. agitation 6. foam
• batch

  low cell debris, low risk of contamination, fair productivity
• fed batch

  sterility, medium cell debris
• perfusion


  high productivity, high cell debris
• cell culture media impacts cell growth and viability, protein production and protein quality by providing nutrients and stimulatory and regulatory effects