What are the advantages of recombinant DNA (rDNA) technology on biopharmaceutical products?
- Overcome limitations regarding source availability
- Natural sources can be rare, expensive, low yield
- Production of safer biopharmaceuticals
- E.g., can eliminate transmission of blood-borne pathogens - when pdt directly isolated from HIV/Hep-B infected sources
- Alternative way to obtain protein-based pdts
- Other than direct extraction from inappropriate source material (e.g., urine, placenta)
- Opportunities to design desirable mutations
- E.g., greater clinical efficacy, greater protein stability for longer shelf-life, shorter or longer circulation half-life
=> cheaper, safer, abundant supply
Briefly describe the upstream process in recombinant protein making
- Host cell transfected with rDNA, each transfected cell diff from each other
- Each transfected cell differ from each other in terms of number of copies of plasmids being transfected => higher the copies of plasmid, higher is the amount of protein being expressed
- Selection of 1 transfected cell that possess the best cell growth properties and highest protein yield => development of master cell line
In upstream manufacturing process, how might quality and safety of the product be compromised?
- Use of E. coli cells
- Use of CHO cells
E. coli:
- Endotoxin lipopolysaccharides (LPS) present in outer membrane of gram-negative bacteria, are pyrogens
- Host cell proteins
CHO cells:
- Host cell proteins
How might downstream manufacturing process help to improve quality and safety of the product?
Protein purification process
- Extraction (lysis)
- Enrichment/isolation of protein of interest (consists removal of cell debris, removal of nucleic acid and lipids, concentration of protein of interest, purification of protein of interest) (methods such as: centrifugation, filtration, chromatography etc.)
- Removal of contaminating substances
*Can also have viral inactivation steps
[BIOSIMILARS]
What is a biosimilar?
Biosimilar is a biologic that is almost identical to the original biologic
Reference to innovator/reference biologic is crucial for its approval by regulatory authorities
[BIOSIMILARS]
Can biosimilars be entirely identical to innovator biologic?
Why?
No, in fact each biological product displays variability even between different batches of the same product.
This is due to variability of the biological expression system and manufacturing process (both upstream and downstream).
- E.g., if product undergoes PTM within host cell, PTM pattern may differ b/w diff batches from diff host cells (e.g., glycosylation pattern can vary - diff types of glycan can be attached to diff AAs, can be straight or branched chain)
[BIOSIMILARS]
What are some factors in the manufacturing process that influence the final characteristics of a biologic?
- Type of host cell (e.g., growth rate of CHO cells)
- Development of genetically modified cell for production
- Cell culture system/conditions (e.g., which nutrient, which growth factors)
- Production process
- Purification process
- Formulation of recombinant protein
[BIOSIMILARS]
Compare the differences in variability between drugs of diff types (chemical drugs, biologics of low MW, biologics of high MW)
Chemical drugs
- Well-characterized production of generics as analytical criteria are based on chemical compositions
Biologics of relatively low MW:
- E.g., insulin, EPO, cytokines, growth factors such as filgrastim
- More straightforward development of biosimilars (small proteins have shorter half-life and more straightforward PK profiles)
Biologics of larger MW:
- E.g., mAbs with PTM - impossible to engineer 100% identical to innovator biologic
- E.g., pattern of glycosylation and amount of glycosylation dependent on manufacturing process
[BIOSIMILARS]
What can be used to access difference in glycosylation pattern?
Mass spectrometry
E.g., can identify linear vs branched chain, that can affect activity and half-life
[BIOSIMILARS]
To identify heterogeneity, the glycans from Fc fragment of IgGs are labelled with fluorescent probes, view peaks on chromatogram.
Describe the peak appearance of polyclonal IgG obtained from pooled plasma of human donors.
Multiple peaks, suggesting highly heterogeneous pattern of glycosylation among polyclonal IgGs
[BIOSIMILARS]
To identify heterogeneity, the glycans from Fc fragment of IgGs are labelled with fluorescent probes, view peaks on chromatogram.
Describe the peak appearance of monoclonal IgG produced by CHO cells.
Less peaks, suggesting less heterogeneous pattern of glycosylation among monoclonal IgGs
[BIOSIMILARS]
How can CHO cells be genetically modified to alter the recombinant IgG to produce defucosylated IgG?
E.g., mutant CHO cells can be modified to lack enzyme that adds fucose to the Fc domain
[BIOSIMILARS]
How does the pattern of glycosylation differ between humans and CHO cells
Sialylated glycans are absent in monoclonal antibodies produced in CHO cells
[BIOSIMILARS]
What are the requirements for approval of biosimilars?
- Extensive in vitro studies demonstrating similarity to the reference/innovator biologic
- Non-clinical and clinical studies demonstrating comparable PK, clinical efficacy, safety, and immunogenicity (studies must have 2 arms - innovator vs biosimilar)
*Note that the methods/assays used to establish comparability of biosimilars to reference product should be sufficiently SPECIFIC and SENSITIVE to detect differences b/w the two products
[BIOSIMILARS]
What are some debatable issues that have arised with the use of biosimilars?
- Can biosimilars serve as automatic substitutes, can they be interchangably used with the innovator?
- Can there be indication extrapolation?
