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Bio cmc development
- 1. BIO-CMC Development China medical university Master program for pharmaceutical manufacture Shuo Yen Hsu
- 2. 2 Cell line development Limiting dilution Lead clones MTX/puromycin selection CHO cell Candidate clones Feeding study & Characterization Stability study Selection
- 3. 3 Feeding study: Viability, VCD, titer, and Qp of the lead clones will be examined Feeding study & Characterization Characterization Item SDS-PAGE (reducing) SDS-PAGE (non-reducing) Western blot analysis Glycan profile analysis by GXII Antigen binding assay or Bioassay Molecular weight by LC-MS Reducing and de-glycosylated Molecular weight by LC-MS Final product clone Candidate clones
- 4. 4 Cell bank establishment Final product clone FRCB 20 vials MCB 200 vials WCB 200 vials Upstream process development GMP manufacture Cell line characterization According to ICH Q5D EPC FRCB: Final Research Cell Bank MCB: Master Cell Bank WCB: Working Cell Bank EPC: End of Production Cell Bank
- 5. ICH Q5D 5 Tests of Identity Morphological analysis may be a useful tool in conjunction with other tests. Isoenzyme analysis is sufficient to confirm the species of origin for cell lines. Other technologies may be substituted to confirm species of origin, for example, banding cytogenetics or use of species-specific antisera. An alternative strategy would be to demonstrate the presence of unique markers, for example, by using DNA analysis to detect a genomic polymorphism pattern.
- 6. ICH Q5D 6 Tests of Purity Tests for the presence of bioburden should be performed on individual containers of the MCB and WCB, the presence of mycoplasma especially should be performed. Virus testing of cell substrates should be designed to detect a wide spectrum of viruses, applicants should consult the ICH Q5A (R1). The purity of cell substrates can be compromised through contamination by cell lines of the same or different species of origin. Cell identity are also considered adequate tests to detect cross-contamination by other cell lines.
- 7. ICH Q5A 7 General testing requirement in compliance with ICH Q5A http://www.biooutsource.com/
- 8. ICH Q5D 8 Cell Substrate Stability For cell lines containing recombinant DNA expression constructs, consistency of the coding sequence should be verified by either nucleic acid testing or product analysis, as described in ICH Q5B. Other specific traits which may include morphological characteristics, growth characteristics, biochemical markers, immunological markers, productivity, or other relevant genotypic or phenotypic markers may be useful. In some case, direct comparison of the MCB with the production cells is difficult, one may compare the characteristics of cells at the initial stages beyond the limit of in vitro cell age, for example, oxygen or glucose consumption rates, ammonia or lactate production rates may be useful for such testing.
- 9. ICH Q5B 9 • Restriction endonuclease mapping should be used to analyse for copy number, insertions or deletions, and the number of integration sites. • For extrachromosomal expression systems, the percent of host cells retaining the expression construct should be determined, and the expression construct should be isolated and the nucleotide sequence should be verified. • The protein coding sequence for the recombinant protein product of the expression construct should be verified. • For cells with chromosomal copies of the expression construct, the nucleotide sequence encoding the product could be verified by recloning and sequencing. • Alternatively, the nucleic acid sequence encoding the product could be verified by sequencing of cDNA clones or material amplified by the polymerase chain reaction.
- 10. 10 Upstream process Cell bank Shake flask 1 L Spinner flask 50 or 200 L bioreactorProduction Harvest Clarification (Filtration system) Concentration (TFF system) Thaw Expansion Expansion Expansion
- 11. 11 Downstream process Virus inactivation Purification column 1 Purification column 2 Purification column 3 DS (Drug substance) Nanofiltration & UF/DF Virus clearance According to ICH Q5A
- 12. ICH Q5A 12 • A combination of factors must be considered when judging the effectiveness of virus inactivation/removal procedures. • The appropriateness of the test viruses used; • The design of the clearance studies; • The log reduction achieved; • The time-dependence of inactivation; • The potential effects of variation in process parameters on virus inactivation/removal; • The limits of assay sensitivities; • The possible selectivity of inactivation/removal procedure(s) for certain classes of viruses. • Effective clearance may be achieved by any of the following: multiple inactivation steps, multiple complementary separation steps, or combinations of inactivation and separation steps. • Well designed separation steps, such as chromatographic procedures, filtration and extractions, can be effective virus removal steps.
- 13. 13 Formulation http://www.wuxibiologics.com/services-solutions/development/formulation-development/
- 14. Excipient selection 14 1. Protein stabilizers • Stabilize the protein native conformation • polyols, sugars, amino acids, amines, and salting out salts 2. Polymers and proteins • Non-specifically to stabilize proteins and enhance protein assembly • dextran, hydroxyl ethyl starch (HETA), PEG-4000, and gelatin 3. Surfactants • Non-ionic surfactants are widely used to stabilize proteins, suppress aggregation, and assist in protein refolding • Tween 80 and Tween 20 4. Amino acids • Stabilize proteins by a variety of mechanisms • histidine, arginine, and glycine 5. Preservatives • Prevent microbial growth • benzyl alcohol, m-cresol, and phenol
- 16. 16 Fill/finishing Aseptic fill Formulation Sterilization depyrogenation Wash Labeling and packaging Appearance inspection Capping DS (Drug substance) DP (Drug product) QA release
- 17. 17 Protein characterization Structure properties Amino acid sequence N-glycosylation site N-glycan structure Disulfide linkage Higher order structure Bioactivity ADCC CDC Proliferation inhibition Specific bioactivity Immunological properties Binding with targets Binding with receptor for immunoactivity Purity and impurities Aggregates Fragments Oxidized form Deamidated form Acidic form Basic form Host cell protein Protein A Host cell derived DNA DS/DP characterization According to ICH Q6B
- 18. ICH Q6B 18 • Physicochemical properties • Determination of the composition, physical properties, and primary structure of the desired product. • Structural heterogeneity occurs in proteins due to the biosynthetic processes used by living organisms, and can be a mixture of anticipated post-translationally modified forms, their presence may have no deleterious effect on the safety and efficacy of the product. • If a consistent pattern of product heterogeneity is demonstrated, an evaluation of the activity, efficacy and safety (including immunogenicity) of individual forms may not be necessary.
- 19. ICH Q6B 19 • Biological activity • Animal-based biological assays, which measure an organism's biological response to the product; • Cell culture-based biological assays, which measure biochemical or physiological response at the cellular level; • Biochemical assays, which measure biological activities such as enzymatic reaction rates or biological responses induced by immunological interactions.
- 20. ICH Q6B 20 • Immunochemical properties • Binding assays of the antibody to purified antigens and defined regions of antigens should be performed, as feasible, to determine affinity, avidity and immunoreactivity (including cross-reactivity). • For some drug substances or drug products, the protein molecule may need to be examined using immunochemical procedures (e.g., ELISA, Western-blot) utilizing antibodies which recognize different epitopes of the protein molecule. • Immunochemical properties of a protein may serve to establish its identity, homogeneity or purity, or serve to quantify it.
- 21. ICH Q6B 21 • Purity, impurities and contaminants • The drug substance include several molecular variants, when variants have properties comparable to the desired product, they are considered product-related substances and not impurities. • Process-related impurities derived from the manufacturing process, i.e., cell substrates (e.g., host cell proteins, host cell DNA), cell culture (e.g., inducers, antibiotics, or media components). • Contaminants in a product include all adventitiously introduced materials, such as chemical and biochemical materials (e.g., microbial proteases), and/or microbial species, and they should be controlled with appropriate in-process acceptance criteria.
- 22. Thank you!