LEC 4 Flashcards
Pharmaceuticals vs biopharmaceuticals
- Pharmaceuticals are smaller molecules.
Biopharmaceuticals are larger molecules. - Pharmaceuticals are produced through chemical synthesis.
Biopharmaceuticals are produced in or extracted from living system/cells - Pharmaceuticals have defined (simpler) structures and easier to characterize.
Biopharmaceuticals have complex structures and more difficult to characterize. - Pharmaceuticals are relatively stable to heat and less prone to microbial contamination.
Biopharmaceuticals are more sensitive to heat and prone to microbial contamination.
Types of biological products
- Traditional Biological Medicinal Product (TBMP)
- Derived and extracted directly from human or animal tissues - Biotechnology-derived Medicinal Product (BDMP)
- Protein products are produced in living system via biotechnology
- 2 methods (Recombination DNA technology & Hybridoma technology)
Examples of biological medicinal products
- Protein
- Sugars
- Nucleic acids
- Hormones
- Vaccines
What is biotechnology?
It is the use of living cells/organisms, or their products to modify human and animal heath, mankind and his environment
In Recombinant DNA technology, what does the recombinant plasmid contains?
- Target gene
- Promoter
- Antibiotic-resistance gene
Bacterial cell + recombinant plasmid
= transformed bacteria cell
Why is myeloma cells used in hybridoma technology?
Myeloma cells are tumor cells with immortality. They can divide and replicate rapidly and in large quantity, leading to increased production of BDMP
Antibody-producing CHO cells + myeloma (tumor) cells
= hybridoma cells (antibody-producing ability & immortality)
Bacteria cell vs Mammalian cells (5)
- Bacteria cells cultivation is faster (~20min/generation), relative straightforward fermentation.
Mammalian cells cultivation is slower (~20-24h/generation), relatively complicated cell culture. - Protein product from bacteria cells are simpler due to the lack of post-translational modification.
Protein product from mammalian cells are more complex with post-translational modification (eg glycosylation) - Bacteria cells store the protein product intracellularly, hence disruption of cell is needed to harvest product.
Mammalian cells secrete the protein product extracellularly, hence disruption of cell is not needed. - There is a lower yield of protein product from bacteria cell due to more difficult purification.
There is a higher yield of protein product from mammalian cell due to less difficult purification. - Bacteria cells produce relatively safe biotechnological product.
Mammalian cells produce biological products with many safety concerns (eg endogenous viruses and carcinogenic residual DNA)
Criteria to use mammalian cells (eg CHO cells) for BDMP
Purification process can reduce carcinogenic residual DNA < 10ng/dose
Critical GMP and QA issues to address (5)
- Genetic stability of host cell with plasmid / vector / gene of interest
- Absence of impurities from nutrient media & starting material
- Assuring quality & yield
- Absence of endogenous (eg herpes virus, EBV & retrovirus) and adventitious viruses
- Elimination of potentially carcinogenic residual DNA
What is a biosimilar product?
A biosimilar product is a biological product that is “highly similar” and has no significant difference from an existing FDA approved reference product (aka innovator product)
Minor differences in clinically-inactive components are acceptable (eg buffers & stabilisers)
Definition of “highly similar”
High degree of similarity in terms of
- Molecular structure & potency (medchem)
- Toxicity
- PK
- PD
- Immunogenicity
Why is biosimilar important?
Reduce price of similarly efficacious treatment to innovator products
Does GMP and quality assurance requirements for innovator product applicable to biosimilar products?
Yes.
Major steps in manufacture of BDMP (7)
- Cell banking
- Cell cultivation
- Harvesting
- Purification
- Viral clearance (viral inactivation & removal)
- Batching & storage of bulk biological API
- Formulation, filling & packaging into final dosage form
Inspection of GMP compliance to be done for all 7 steps
Types of cell banks (2)
- Master Cell Bank (MCB)
- Contains well-characterised cells derived from specific cell line
- more tests conducted on MCB - Working Cell Bank (WCB)
- Less tests are done for WCB before use for manufacture
- derived from one or more containers of MCB
Inspection of Cell Bank system (step1) (3)
- Documentation of cell origin & history
- Management of cell banks
- Contract with outsourced testing laboratories (cos need expertise)
Cell cultivation definition
Refers to both microbial fermentation and mammalian cell culturing
Upstream processes of manufacturing process
- Cell banking
- Cell expansion & scale up
- Cultivation
- Harvesting
Downstream processes of manufacturing process
- Primary purification (affinity chromatography)
- Viral inactivation (low pH/detergent)
- Secondary purification (column chromatography)
- Viral removal (nanofiltration)
- Batching & storage of bulk API
- Formulation, filling, packaging & QC of finished biological product
Elemental requirement for bacterial fermentation
CNPS - Carbon - Nitrogen - Phosphorus - Sulfur Some trace elements
Types of Fermentations (3)
- Batch fermentation
- Oxygen is continuously supplied
+ve : minimal risk of contamination
-ve : low yield due to decline phase as nutrients depletes (not economical) - Continuous fermentation
- Log phase (steady growth rate)
+ve : high yield
-ve : high contamination risk due to open system - Batch-fed fermentation (more commonly used today)
- Semi-closed system
- Nutrients are added while waste products are removed at periodic intervals
Types of Cell Culturing (2)
- Anchored system
- Suspension system
- No lag, log, stationary and decline phases
- Minor differences in nutrients have MAJOR effect on cell growth & protein production
- Optimal shearing, stirring speed and circulation are crucial parameters since mammalian cells are fragile (lack rigid cell wall)
- Protein products are secreted extracellularly into the media
Nutrient media used in Cell Culturing
- Initially used Bovine Serum but reduced due to concerns of BSE prions (mad cow disease)
- Today, move towards the use of serum-free nutrient media for safety & cost concerns
Inspection of Cultivation process (step2) (3)
- Control of starting materials (cell lines, nutrient media, water etc)
- Control of cell culture conditions
- Maintenance & cleaning of fermenters/bioreactors (now SUT)
SUT
Single Use Technology
Methods for cell disruption (cell lysis)
- Non-mechanical
- Surfactants
- Lysozyme
- Osmotic shock
- Alkali
- Solvents - Mechanical
- Milling
- Ultrasonification
- Homogenization
- Oscillation
Challenges of cell disruption (cell lysis) (4)
- Oxidation of protein product
- Heat denaturation of protein product
- Uncontrolled release of cell debris
- Degradation of protein product by lysosomes released during cell lysis
Definition of chromatography
The separation of different molecules in a mixture while moving in a mobile phase
Affinity chromatography
The protein product interacts or bond specifically with the stationary phase.
The bound protein is subject to further purification.
Inspection of Harvesting process (step 3)
- Control of chromatographic columns, buffers & other materials
- Level of purity relating to usage of product (chronic vs acute/single use)
Viral inactivation methods (2)
- Chemical :
- Low pH
- Surfactants / detergents - Physical :
- Heat
- UV
Viral removal methods (4)
- Precipitation (ammonium sulfate)
- Membrane filtration
- Nanofiltration
- Column chromatography
Why need validation of viral clearance? (4)
- No single test can demonstrate the presence of all known viruses
- Sensitivity of test system require a minimum level of viral contamination to record a positive result
- Tests are limited by statistical consideration in sampling (need representative sample)
- Virus only identified many years after the product manufacture
Criteria to ensure product is free from viral contamination
- Viral testing
2. Validation of manufacturing processes that inactivate & remove viruses
Aim of viral clearance study / validation
To demonstrate that manufacturing or purification processes can eliminate substantially more viruses than what may potentially be present in the unprocessed bulk
To obtain reasonable assurance that product is free from viral contamination
Virus model / panel for viral clearance studies
- Enveloped, DNA
- Enveloped, RNA
- Non-enveloped, DNA
- Non-enveloped, RNA
Test for viruses of varying physicochemical properties, sizes and chemical resistance to demonstrate robustness of viral clearance capability
Definition of a batch
Batch means a specific quantity of drug or other material that is intended to have uniform character and quality, within specified limits, and is produced according to a single manufacturing order during the same cycle of manufacture
Can biological products be manufactured in sub-lots then pooled together as a batch?
Yes
Inspection of batching & storage of bulk (step6)
Batching :
- Containers of biological API
- Stability of biological API in containers
- Compatibility of biological API & excipients
Storage :
- Storage conditions of biological API
- Size of storage space of biological API
- Security of area
Inspection of Formulation, Filling, Packaging & QC into finished product
- Absence of endogenous & adventitious viruses
- Elimination of residual DNA
- Pyrogen tests
- Modified 21 CFR method (test for mycoplasma contamination)
Repeats vs reproducibility
Repeats – using same batch of materials & other factors
Reproducibility – using different batch of materials & other factors (eg days, technicians)
Precision vs accuracy
Precision – closeness of agreement among measurements
Accuracy – degree of closeness to the true value
Specificity vs sensitivity
Specificity – ability to assess unequivocally the analyte in the presence of expected components
Sensitivity – lower limit of detection
Why was the use of mammalian cells not used earlier?
Due to the possible contamination of potentially oncogenic DNA in the protein product
Mean Generation Time (MGT) for
- bacterial cells
- CHO cells
- humans
Bacterial cells - 20min (binary fission)
CHO cells - 20-24h (mitosis)
Humans - 20-25 years
Robustness
Ability of the method to deliver accurate and precise results under normal operating conditions
