Biologics Production and Contamination Quiz

Protein Denaturation and Renaturation

• Denaturation can be good, as it allows misfolded proteins in bacteria to be further denatured and then renatured.
• Glycosylation
• Virus
• Pirogens can be contaminants in eukaryotic cells.
• Recombinant proteins in E. coli.
• E. coli is a common host for producing heterologous proteins.
• Glycosylation is a fundamental post-translational process
• Bacteria can't modify proteins with glycans, eukaryotes can.
• Different glycans attached to a protein creates variation
• DNA is messenger RNA!

Protein Expression in E. coli

• Many reviews exist on recombinant protein production in bacteria
• Protein production is important for drug discovery and development.
• Genetically engineered yeast strains allow for mammalian-like glycosylation patterns.
• CHO and NSO cells are commonly used for mAb production.

Glycosylation

• Glycosylation is a fundamental post-translational process.
• Bacteria can't perform glycosylation.
• Eukaryotes can modify proteins with glycans, leading to diverse protein variations.

Sialic Acid

• Sialic acid
• Important for the serum half-life of glycoproteins
• Prevents degradation and removal from the blood.
• Prevents antigenic determinants from exposing glycoproteins.
• Influencing the thermal stability, its resistance to proteolytic degradation and its solubility.

Industrial Bioprocessing of Biologics

• Cell harvesting and culture
• Microbial purity is important for prokaryotic systems.
• Virus-free systems are important for mammalian cell cultures, which often utilize viral removal steps.
• Typical cell culture production schemes range from small-scale T-flasks to large-scale bioreactors.
• Fed-batch fermentation is a method to extend fermentation time due to added nutrients.
• Continuous fermentation allows for continuous product extraction, but introduces risk of contamination.

Culture Media and Other Materials

• Media quality and traceability are important in bioprocessing.
• Contaminants in the media can arise from formulation or substances used.
• Extraction and purification of resulting products depend on used cells and proteins.

Process Characterization

• The process must be designed to be suitable for routine commercial manufacturing.
• Process development is the stage to reach a potential process design.
• Process evaluation involves performing steps on a small and/or commercial scale.
• Process verification studies are to confirm manufacturing process effectiveness.

Downstream Processing

• Four main sections: primary recovery, viral clearance, purification, and formulation.
• Primary recovery involves removing contaminating components.
• Viral Clearance is to remove viruses from mammalian cell-derived proteins.
• Purification ensures that the product meets purity specifications.
• Formulation is to mix the product with appropriate excipients.

Important Concepts in Bioprocessing

• Mammalian cell cultures are often grown in stirred tank bioreactors.
• Sterilization methods (physical and chemical) are crucial to eliminating contaminants.
• Process characterization involves designing processes suitable for commercial manufacturing.
• Process validation confirms efficiency and consistency.

Cell Culture Process Design

• Robust mammalian cell lines with high expression capabilities and large-scale continuous stirred tank reactor (CSTR) formats are common and preferred.
• High throughput and process-oriented solutions (scale-up, cleaning, sterilization) are required.
• Cell culture process design, robust and reproducible mammalian cell culture processes, suspension, and/or batch processes.
• Gene-amplification and host cell protein removal steps are important.

Cell Line Transfection and Selection

• CHO cells are the most utilized mammalian host cells.
• CHO, NSO, BHK, HEK-293, and PER-C6 are alternative cell lines.
• These cells are adapted for suspension culture.
• Virus removal is common in these systems.

Additional Notes on the topic

• The use of DHFR strains and MTX induced gene amplification are important aspects for amplification of the target gene.
• Upstream processes can be optimized using high expression vector systems, cell lines capable of high product output per day, optimized media, quality of instrumentation and process conditions.
• Crucial factors for cell culture optimization include, but are not limited to, cell and physical parameters, biochemical parameters, processes and technology.