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.
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
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.
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
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.
